Leukemia associated genes

ABSTRACT

The invention describes leukemia associated genes, including fragments and biologically functional variants thereof. Also included are polypeptides and fragments thereof encoded by such genes, and antibodies relating thereto. Methods and products also are provided for diagnosing and treating conditions characterized by expression of a preproTRH, tryptase-L and/or Oct-T1 gene product.

FIELD OF THE INVENTION

This invention relates to nucleic acid molecules and encodedpolypeptides which are expressed preferentially in leukemia. The nucleicacid molecules and encoded polypeptides are useful in, inter alia,diagnostic and therapeutic contexts.

BACKGROUND OF THE INVENTION

The phenotypic changes which distinguish a tumor cell from its normalcounterpart are often the result of one or more changes to the genome ofthe cell. The genes which are expressed in tumor cells, but not innormal counterparts, can be termed "tumor specific" genes. These tumorspecific genes are markers for the tumor phenotype. The expression oftumor specific genes can also be an essential event in the process oftumorigenesis.

Typically, the host recognizes as foreign the tumor specific genes whichare not expressed in normal non-tumorigenic cells. Thus, the expressionof tumor specific genes can provoke an immune response against the tumorcells by the host. Tumor specific genes can also be expressed in normalcells within certain tissues without provoking an immune response. Insuch tissues, expression of the gene and/or presentation of anordinarily immunologically recognizable fragment of the protein producton the cell surface may not provoke an immune response because theimmune system does not "see" the cells inside these immunologicallyprivileged tissues. Examples of immunologically privileged tissuesinclude brain and testis

The discovery of tumor specific expression of a gene provides a means ofidentifying a cell as a tumor cell. Diagnostic compounds can be based onthe tumor specific gene, and used to determine the presence and locationof tumor cells. Further, when the tumor specific gene contributes to anaspect of the tumor phenotype (e.g., unregulated growth or metastasis),the tumor specific gene can be used to provide therapeutics such asantisense nucleic acids which can reduce or substantially eliminateexpression of that gene, thereby reducing or substantially eliminatingthe phenotypic aspect which depends on the expression of the particulartumor specific gene.

As previously noted, the polypeptide products of tumor specific genescan be the targets for host immune surveillance and provoke selectionand expansion of one or more clones of cytotoxic T lymphocytes specificfor the tumor specific gene product. Examples of this phenomenon includeproteins and fragments thereof encoded by the MAGE family of genes, thetyrosinase gene, the Melan-A gene, the BAGE gene, the GAGE gene, theRAGE family of genes, the PRAME gene and the brain glycogenphosphorylase gene, as are detailed below. Thus, tumor specificexpression of genes suggests that such genes can encode proteins whichwill be recognized by the immune system as foreign and thus provide atarget for tumor rejection. Such genes encode "tumor rejection antigenprecursors", or TRAPs, which may be used to generate therapeutics forenhancement of the immune system response to tumors expressing suchgenes and proteins.

The process by which the mammalian immune system recognizes and reactsto foreign or alien materials is a complex one. An important facet ofthe system is the T cell response. This response requires that T cellsrecognize and interact with complexes of cell surface molecules,referred to as human leukocyte antigens ("HLA"), or majorhistocompatibility complexes ("MHCs"), and peptides. The peptides arederived from larger molecules which are processed by the cells whichalso present the HLA/MHC molecule. See in this regard Male et al.,Advanced Immunology (J.P. Lipincott Company, 1987), especially chapters6-10. The interaction of T cells and complexes of HLA/peptide isrestricted, requiring a T cell specific for a particular combination ofan HLA molecule and a peptide. If a specific T cell is not present,there is no T cell response even if its partner complex is present.Similarly, there is no response if the specific complex is absent, butthe T cell is present. The mechanism is involved in the immune system'sresponse to foreign materials, in autoimnmune pathologies, and inresponses to cellular abnormalities. Much work has focused on themechanisms by which proteins are processed into the HLA bindingpeptides. See, in this regard, Barinaga, Science 257: 880, 1992; Fremontet al., Science 257: 919, 1992; Matsumura et al., Science 257: 927,1992; Latron et al., Science 257: 964, 1992.

The mechanism by which T cells recognize cellular abnormalities has alsobeen implicated in cancer. For example, in PCT applicationPCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, andincorporated by reference, a family of genes is disclosed, which areprocessed into peptides which, in turn, are expressed on cell surfaces,which can lead to lysis of the tumor cells by specific CTLs. The genesare said to code for "tumor rejection antigen precursors" or "TRAP"molecules, and the peptides derived therefrom are referred to as "tumorrejection antigens" or "TRAs". See Traversari et al., J. Exp. Med.176:1453-1457, 1992; van der Bruggen et al., Science 254: 1643,1991; DePlaen et al., Immunogenetics 40:360-369, 1994 for further information onthis family of genes. Also, see U.S. patent application Ser. No.807,043, filed Dec. 12, 1991, now U.S. Pat. No. 5,342,774.

In U.S. patent application Ser. No. 938,334, now U.S. Pat. No.5,405,940, the disclosure of which is incorporated by reference,nonapeptides are taught which are presented by the HLA-A1 molecule. Thereference teaches that given the known specificity of particularpeptides for particular HLA molecules, one should expect a particularpeptide to bind one HLA molecule, but not others. This is important,because different individuals possess different HLA phenotypes. As aresult, while identification of a particular peptide as being a partnerfor a specific HLA molecule has diagnostic and therapeuticramifications, these are only relevant for individuals with thatparticular HLA phenotype. There is a need for further work in the area,because cellular abnormalities are not restricted to one particular HLAphenotype, and targeted therapy requires some knowledge of the phenotypeof the abnormal cells at issue.

In U.S. patent application Ser. No. 008,446, filed Jan. 22, 1993 andincorporated by reference, the fact that the MAGE-1 expression productis processed to a second TRA is disclosed. This second TRA is presentedby HLA-Cw16 molecules, also known as HLA-C*1601. The disclosure showsthat a given TRAP can yield a plurality of TRAs.

In U.S. patent application Ser. No. 994,928, filed Dec. 22, 1992, andincorporated by reference herein, tyrosinase is described as a tumorrejection antigen precursor. This reference discloses that a moleculewhich is produced by some normal cells (e.g., melanocytes), is processedin tumor cells to yield a tumor rejection antigen that is presented byHLA-A2 molecules.

In U.S. patent application Ser. No. 08/032,978, now U.S. Pat. No.5,620,886, and incorporated herein by reference in its entirety, asecond TRA, not derived from tyrosinase is taught to be presented byHLA-A2 molecules. The TRA is derived from a TRAP, but is coded for by aknown MAGE gene. This disclosure shows that a particular HLA moleculemay present TRAs derived from different sources.

In U.S. patent application Ser. No. 079,110, now U.S. Pat. No. 5,571,711and entitled "Isolated Nucleic Acid Molecules Coding For BAGE TumorRejection Antigen Precursors" and Ser. No. 196,630, filed Feb. 15, 1994,and entitled "Isolated Peptides Which form Complexes with MHC MoleculeHLA-C-Clone 10 and Uses Thereof" the entire disclosures of which areincorporated herein by reference, an unrelated tumor rejection antigenprecursor, the so-called "BAGE" precursor, is described. TRAs arederived from the TRAP and also are described. They form complexes withMHC molecule HLA-C-Clone 10.

In U.S. patent application Ser. No. 096,039, filed Jul. 22, 1993 andentitled "Isolated Nucleic Acid Molecules Coding for GAGE TumorRejection Antigen Precursors" and Ser. No. 250,162, now U.S. Pat. No.5,610,013 and entitled "Method for Diagnosing a Disorder by DeterminingExpression of GAGE Tumor Rejection Antigen Precursors", the entiredisclosures of which are incorporated herein by reference, anotherunrelated tumor rejection antigen precursor, the so-called "GAGE"precursor, is described. The GAGE precursor is not related to the BAGEor the MAGE family.

In U.S. patent application Ser. No. 08/408,015, filed Mar. 21, 1995, andentitled "RAGE Tumor Rejection Antigen Precursors", incorporated hereinby reference in its entirety, another TRAP is taught which is notderived from any of the foregoing genes. The TRAP is referred to asRAGE. In U.S. patent application Ser. No. 08/530,015, filed Sep. 20,1995, and entitled "Isolated RAGE-1 Derived Peptides Which Complex withHLA-B7 Molecules and Uses Thereof", also incorporated by reference, theTRA derived form one member of the RAGE family of genes is taught to bepresented by HLA-B7 molecules. This disclosure shows that additionalTRAPs and TRAs can be derived from different sources.

In U.S. patent application Ser. no. 08/253,503, now U.S. Pat.No.5,589,334, and entitled "Isolated Nucleic Acid Molecule Which Codesfor a Tumor Rejection Antigen Precursor Which is Processed to an AntigenPresented by HLA-B44", incorporated herein by reference in its entirety,another TRAP is taught which is not derived from any of the foregoinggenes. The gene encoding the TRAP is referred to as MUM-1. A tumorrejection antigen, LB-33B, is described in the application.

In U.S. patent application Ser. No. 08/373,636, filed Jan. 17, 1995, andentitled "Isolated Nucleic Acid Molecule Which Codes for a TumorRejection Antigen Precursor Which is Processed to Antigens Presented byHLA Molecules and Uses Thereof", incorporated herein by reference in itsentirety, other TRAPs are taught which are derived from LB33 andpresented by HLA-B13, HLA-Cw6, HLA-A28 and HLA-A24.

In PCT publication WO96/10577, published Apr. 11, 1996, and entitled"Isolated Nucleic Acid Molecule Coding for a Tumor Rejection AntigenPrecursor DAGE and Uses Thereof", incorporated herein by reference inits entirety, another TRAP is taught which is not derived from any ofthe foregoing genes. The TRAP was referred to as DAGE, but is nowreferred to as PRAME. A tumor rejection antigen is described in theapplication which is presented by HLA-A24.

In U.S. patent application Ser. No. 08/487,135, filed Jun. 7, 1995, andentitled "Isolated Nucleic Acid Molecule, Peptides Which Form Complexeswith MHC Molecule HLA-A2 and Uses Thereof", incorporated herein byreference in its entirety, another TRAP is taught which is not derivedfrom any of the foregoing genes. The TRAP is referred to as NAG. VariousTRAs derived from NAG and presented by HLA-A2 are taught in thisapplication.

In U.S. patent application Ser. No. 08/403,388, now U.S. Pat. No.5,587,289, and entitled "Isolated Nucleic Acid Molecules Which AreMembers of the MAGE-Xp Family and Uses Thereof", incorporated herein byreference in its entirety, three TRAPs are taught which are not derivedfrom any of the foregoing genes. These TRAPs are referred to asMAGE-Xp2, MAGE-Xp3 and MAGE-Xp4.

The work which is presented by the papers, patents and patentapplications described above deal, for the most part, with the MAGEfamily of genes, the BAGE gene, the GAGE gene and the RAGE family ofgenes.

In U.S. patent application Ser. No. 08/672,351, filed Jun. 25, 1996, andentitled "Brain Glycogen Phosphorylase Cancer Antigen", incorporatedherein by reference in its entirety, another TRAP is taught which is notderived from any of the foregoing genes. This TRAP is a gene which isexpressed normally in the brain and retinal pigmented epithelium. It wasdiscovered that the brain glycogen phosphorylase gene is expressed inmelanoma cells, and encodes tumor rejection antigens and precursorsthereof. It now has been discovered that additional genes similarly areexpressed in a tumor associated pattern in leukemia cells.

These three genes which are believed to encode tumor rejection antigenprecursors are referred to generally as leukemia associated genes. Thesegenes do not show homology to the MAGE family of genes, to the BAGEgene, the GAGE gene, the RAGE family of genes, the LB33/MUM-1 gene, theNAG gene, the MAGE-Xp family of genes or the brain glycogenphosphorylase gene. Two of the genes are known genes which were notpreviously known to be expressed in a leukemia associated manner. One ofthe genes is an unknown gene. Thus the invention relates to the genesexpressed specifically in certain leukemia cells, tumor rejectionantigen precursors encoded by such genes, as well as related moleculesand applications of these various entities.

The invention is elaborated upon further in the disclosure whichfollows.

SUMMARY OF THE INVENTION

The invention provides isolated nucleic acid molecules encoding leukemiaassociated polypeptides. The invention also provides expression vectorscontaining those molecules and host cells transfected with thosemolecules, as well as isolated polypeptides encoded by the leukemiaassociated nucleic acid molecules and fragments of the isolatedpolypeptides. The foregoing isolated nucleic acid molecules andpolypeptides can be used in the diagnosis or treatment of conditionscharacterized by the expression of a leukemia associated gene.

According to one aspect of the invention, methods for diagnosing adisorder characterized by the expression of a leukemia associatednucleic acid molecule or a leukemia associated polypeptide are provided.The methods involve contacting a biological sample isolated from asubject with an agent that is specific for the leukemia associatednucleic acid molecule or an expression product thereof. In certainembodiments, the leukemia associated nucleic acid molecule hybridizesunder stringent conditions to a molecule having a nucleotide sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, and SEQ ID NO:7. In these certain embodiments, the leukemiaassociated nucleic acid optionally codes for a leukemia associatedpolypeptide. In other embodiments, the agent is a binding agent whichselectively binds to a leukemia associated polypeptide, such as anantibody, cytotoxic T lymphocyte, polypeptide, and the like. The methodsfurther involve determining the interaction or binding between the agentand the nucleic acid molecule or expression product thereof as adetermination of the disorder. In preferred embodiments, the agent is anucleic acid molecule comprising a molecule having a nucleotide sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, fragments thereof, and complements thereof. Incertain embodiments, the interaction between the agent and the nucleicacid molecule is determined by amplifying at least a portion of thenucleic acid molecule. In other preferred embodiments, the leukemiaassociated polypeptide comprises a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8 and fragments thereof. In particularlypreferred embodiments, the agent which binds the leukemia associatedpolypeptide is an antibody. In the foregoing embodiments, the biologicalsample preferably is isolated from a non-fetal-brain tissue, anon-mastocyte tissue, or a non-fetal-testis tissue. In certain of theforegoing embodiments, the leukemia associated nucleic acids andpolypeptides are fragments of the foregoing sequences.

The recognition that peptides derived from leukemia associatedpolypeptides may be presented by HLA molecules and recognized by CTLspermits diagnosis of certain disorders. Thus, according to anotheraspect of the invention, a method for diagnosis of a disordercharacterized by expression of a tumor rejection antigen derived from aleukemia associated polypeptide is provided. The method involvescontacting a biological sample isolated from a subject with an agentthat is specific for the tumor rejection antigen derived from a leukemiaassociated polypeptide. The method then provides for determining theinteraction between the agent and the tumor rejection antigen derivedfrom a leukemia associated polypeptide as a determination of thedisorder. In certain embodiments, the tumor rejection antigen derivedfrom a leukemia associated polypeptide comprises the amino acid sequenceof a fragment of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.In preferred embodiments, the tumor rejection antigen comprises between7 and 100 consecutive amino acids of the foregoing sequences.Preferably, the biological sample is isolated from non-fetal-brain,non-mastocyte or non-fetal testis tissue. In certain embodiments, theagent is an antibody.

The above-described method provides diagnosis of a disorder based on thepresence of leukemia associated TRAs. Another aspect of the inventionprovides methods for diagnosing a disorder characterized by theexpression of a tumor rejection antigen derived from a leukemiaassociated polypeptide which forms a complex with HLA molecules. Themethod involves contacting a biological sample isolated from a subjectwith an agent that binds the complex and then determining bindingbetween the complex and the agent as a determination of the disorder. Inone embodiment, the tumor rejection antigen derived from a leukemiaassociated polypeptide is a peptide comprising the amino acids of afragment of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8. Inpreferred embodiments, the tumor rejection antigen comprises between 7and 100 consecutive amino acids of the foregoing sequences. Preferably,the biological sample is isolated from non-fetal-brain, non-mastocyte ornon-fetal testis tissue. In certain embodiments, the agent is anantibody.

In addition to diagnosis of disorders, treatment of certain disorders isalso desirable. According to another aspect of the invention, methodsfor treating a subject with a disorder characterized by expression of aleukemia associated nucleic acid or polypeptide is provided. The methodinvolves administering to the subject an agent which reduces theexpression of the leukemia associated nucleic acid or polypeptide toameliorate the disorder. The agent is administered in an effectiveamount. In certain embodiments, the leukemia associated nucleic acid orpolypeptide is a tumor rejection antigen and the method involvesadministering to the subject an amount of an agent which enrichesselectively in the subject the presence of complexes of HLA and a tumorrejection antigen derived from a leukemia associated polypeptide encodedby a nucleic acid molecule selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, sufficient to amelioratethe disorder. Preferably, the tumor rejection antigen derived from aleukemia associated polypeptide is a peptide derived from thepolypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.Another method involves administering to a subject in need of suchtreatment an amount of autologous cytolytic T cells sufficient toameliorate the disorder, wherein the autologous cytolytic T cells arespecific for complexes of an HLA molecule and a tumor rejection antigenderived from a leukemia associated polypeptide. Preferably the complexesare formed of HLA and the certain leukemia associated peptides asdescribed above. In other embodiments, the leukemia associated nucleicacid or polypeptide is a nucleic acid and the agent is an antisensenucleic acid. The antisense nucleic acid preferably hybridizes to aleukemia associated nucleic acid selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and fragmentsthereof.

According to another aspect of the invention, a composition is provided.The composition comprises an antisense nucleic acid which binds to aleukemia associated nucleic acid selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and fragmentsthereof. The antisense nucleic acid reduces the expression of theleukemia associated nucleic acid. The composition also includes apharmaceutically acceptable carrier.

The invention in another aspect involves a kit for detecting thepresence of the expression of a leukemia associated polypeptideprecursor. Such kits employ two or more of the above-described nucleicacid molecules isolated in separate containers and packaged in a singlepackage. In one such kit, a pair of isolated nucleic acid molecules isprovided, each of the pair consisting essentially of a molecule selectedfrom the group consisting of a 12-32 nucleotide contiguous segment ofSEQ ID NO:1 and complements thereof, a 12-32 nucleotide contiguoussegment of SEQ ID NO:3 and complements thereof, a 12-32 nucleotidecontiguous segment of SEQ ID NO:5, a 12-32 nucleotide contiguous segmentof SEQ ID NO:7 and complements thereof, and wherein the contiguoussegments are nonoverlapping. Preferably, the pair of isolated nucleicacid molecules is constructed and arranged to selectively amplify atleast a portion of an isolated nucleic acid molecule which hybridizesunder stringent conditions to a molecule selected from the groupconsisting of the nucleic acid sequence of SEQ ID NO:1, the nucleic acidsequence of SEQ ID NO:3, the nucleic acid sequence of SEQ ID NO:5, thenucleic acid sequence of SEQ ID NO:7, nucleic acid molecules whichdiffer from the above in codon sequence due to the degeneracy of thegenetic code and complements thereof. In certain embodiments, the pairof isolated nucleic acid molecules is PCR primers. Preferably one of theprimers is a contiguous segment of SEQ ID NO:1 and another of theprimers is a complement of another contiguous segment of SEQ ID NO:1. Inother preferred embodiments, one of the primers is a contiguous segmentof SEQ ID NO:3 and another of the primers is the complement of anothercontiguous segment of SEQ ID NO:3. In still other preferred embodiments,one of the primers is a contiguous segment of SEQ ID NO:5 and another ofthe primers is the complement of another contiguous segment of SEQ IDNO:5. In yet other preferred embodiments, one of the primers is acontiguous segment of SEQ ID NO:7 and another of the primers is thecomplement of another contiguous segment of SEQ ID NO:7.

The invention in another aspect also provides pharmaceuticalpreparations containing the agents and/or cells of the precedingparagraphs. In one embodiment, the preparation contains apharmaceutically effective amount of preproTRH, tryptase-L, Oct-T1 or afragment thereof that binds an HLA molecule along with pharmaceuticallyacceptable diluents, carriers or excipients. In another embodiment, thepreparation contains a pharmaceutically effective amount of isolatedautologous cytolytic T cells specific for complexes of an HLA moleculeand a tumor rejection antigen derived from preproTRH, tryptase-L orOct-T1.

According to another aspect of the invention, the use of isolatedpreproTRH, tryptase-L, Oct-T1 or fragments thereof in the manufacture ofa medicament is provided. Preferred fragments of the preproTRH,tryptase-L and Oct-T1 molecules are described above. The use ofantisense nucleic acids which bind to a leukemia associated nucleic acidin the manufacture of a medicament is also provided. In certainembodiments, the medicament is an injectable medicament, an oralmedicament, or an inhalable medicament.

According to another aspect of the invention, the use of isolatedpreproTRH, tryptase-L, Oct-T1 or fragments thereof, including antisensenucleic acids, in the manufacture of a medicament for the treatment ofcancer is provided.

According to still another aspect of the invention, an isolated nucleicacid molecule is provided. The molecule hybridizes under stringentconditions to a nucleic acid having a nucleotide sequence selected fromthe group consisting of SEQ ID NO:3 or SEQ ID NO:5. The isolated nucleicacid molecule is a leukemia associated polypeptide precursor and codesfor a tryptase-L leukemia associated polypeptide. The invention furtherembraces nucleic acid molecules that differ from the foregoing isolatednucleic acid molecules in codon sequence to the degeneracy of thegenetic code. The invention also embraces complements of the foregoingnucleic acids. In preferred embodiments, the isolated nucleic acidmolecule comprises the nucleic acid sequence of SEQ ID NO:3 or SEQ IDNO:5. In particularly preferred embodiments, the isolated nucleic acidmolecule comprises the coding region of the foregoing nucleic acids.

According to another aspect of the invention, an isolated nucleic acidmolecule is provided which comprises a molecule selected from the groupconsisting of a unique fragment of nucleotides 487-1499 of SEQ ID NO:3or SEQ ID NO:5 between 12 and 1012 nucleotides in length, a uniquefragment of nucleotides 1665-1774 of SEQ ID NO:5 between 12 and 109nucleotides in length and complements thereof. In preferred embodiments,the unique fragment is at least 14, 15, 16, 17, 18, 20 or 22 contiguousnucleotides of the foregoing. In another embodiment, the isolatednucleic acid molecule consists of between 12 and 32 contiguousnucleotides of the foregoing.

According to yet another aspect of the invention, the invention involvesexpression vectors, and host cells transformed or transfected with suchexpression vectors, comprising the nucleic acid molecules describedabove. The expression vectors optionally include a nucleic acid moleculewhich codes for an HLA molecule. Of course, an HLA-encoding nucleic acidmolecule can also be contained in a separate expression vector. Hostcells transformed or transfected with the foregoing expression vectorsare also provided.

According to another aspect of the invention, an isolated tryptase-Lpolypeptide is provided which is encoded by a nucleic acid moleculewhich hybridizes under stringent conditions to a molecule having thenucleic acid sequence of SEQ ID NO:3, the nucleic acid sequence of SEQID NO:5, nucleic acid molecules which vary from the foregoing accordingto the degeneracy of the genetic code, and complements of any of theforegoing nucleic acid molecules.

According to yet another aspect of the invention, an isolatedpolypeptide is provided which comprises a unique fragment of SEQ ID NO:4or SEQ ID NO:6 between 9 and 189 amino acids in length. Preferably, theunique fragment of the isolated polypeptide binds to apolypeptide-binding agent. In other preferred embodiments, the uniquefragment of the isolated polypeptide binds to an antibody or a cytotoxicT lymphocyte.

The invention also provides isolated polypeptides which selectively binda tryptase-L protein or fragments thereof. Isolated binding polypeptidesinclude antibodies and fragments of antibodies (e.g., Fab, F(ab)₂, Fdand antibody fragments which include a CDR3 region which bindsselectively to the tryptase-L proteins of the invention). The isolatedbinding polypeptides include monoclonal antibodies.

In connection with any of the isolated nucleic acids encoding a leukemiaassociated polypeptide as described above, especially a tumor rejectionantigen derived from a leukemia associated polypeptide, the inventionalso embraces degenerate nucleic acids that differ from the isolatednucleic acid in codon sequence only due to the degeneracy of the geneticcode or complements of any of the foregoing nucleic acids.

The invention also embraces functional variants and equivalents of allof the molecules described above.

These and other objects of the invention will be described in fuirtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing which depicts the sequencing of preproTRH.

FIG. 2 is a schematic drawing which depicts the sequencing of tryptase-Lclones.

FIG. 3 is a schematic drawing which depicts the relation of tryptase-Lclones to the tryptase I gene.

FIG. 4 is a schematic drawing which depicts the Oct-T1/SIAX DP2-64 cDNAclones.

FIG. 5 is a schematic drawing which depicts the CHM 329-2/15 clone ofOct-T1/SIAX DP2-64.

FIG. 6 is a schematic drawing which depicts the sequencing of theCHM363/5 clone of Oct-T1/SIAX DP2-64.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of the preproTRH gene.

SEQ ID NO:2 is the amino acid sequence of the polypeptide encoded by thepreproTRH gene.

SEQ ID NO:3 is the nucleotide sequence of the tryptase-L gene cloneNVB352/1.

SEQ ID NO:4 is the amino acid sequence of the polypeptide encoded by thetryptase-L gene clone NVB352/1.

SEQ ID NO:5 is the nucleotide sequence of the tryptase-L gene cloneNVB352/3.

SEQ ID NO:6 is the amino acid sequence of the polypeptide encoded by thetryptase-L gene clone NVB352/3.

SEQ ID NO:7 is the nucleotide sequence of the SIAX DP2-64 (Oct-T1) gene.

SEQ ID NO:8 is the amino acid sequence of the polypeptide encoded by theSIAX DP2-64 (Oct-T1) gene.

SEQ ID NO:9 is a sense primer for specific PCR amplification ofpreproTRH.

SEQ ID NO:10 is an antisense primer for specific PCR amplification ofpreproTRH.

SEQ ID NO:11 is a sense primer for specific PCR amplification oftryptase-L.

SEQ ID NO:12 is an antisense primer for specific PCR amplification oftryptase-L.

SEQ ID NO:13 is a sense primer for specific PCR amplification of SIAXDP2-64 (Oct-T1).

SEQ ID NO:14 is an antisense primer for specific PCR amplification ofSIAX DP2-64 (Oct-T1).

SEQ ID NO:15 is a sense primer for specific PCR amplification ofβ-actin.

SEQ ID NO:16 is an antisense primer for specific PCR amplification ofβ-actin.

DETAILED DESCRIPTION OF THE INVENTION

The examples which follow show the isolation of nucleic acid moleculeswhich code for polypeptides and are expressed preferentially inmalignant hemopathies, i.e. which are leukemia associated genes. Theseisolated nucleic acid molecules include nucleic acid molecules whichencode preproTRH, tryptase-L and Oct-T1. In particular, the tryptase-Lnucleic acids are different from previously disclosed tryptase codingsequences described supra. Hence, one aspect of the invention is anisolated nucleic acid molecule which includes all or a unique portion ofthe nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:5. Thissequence, and the other leukemia associated gene sequences do not encodea previously recognized tumor rejection antigen precursor, such as aMAGE, BAGE, GAGE, RAGE, LB33/MUM-1, PRAME, NAG, MAGE-Xp or brainglycogen phosphorylase sequence, as will be seen by comparing them tothe sequence of any of the genes described in the references.

The invention thus involves in one aspect preproTRH, tryptase-L andOct-T1 nucleic acids, encoded polypeptides, functional modifications andvariants of the foregoing, useful fragments of the foregoing, as well astherapeutics and diagnostics related thereto.

According to one aspect of the invention, methods for diagnosing adisorder that is characterized by expression of a leukemia associatednucleic acid or polypeptide are provided. The methods involve contactinga biological sample isolated from a subject with an agent specific forthe leukemia associated nucleic acid or polypeptide to detect thepresence of the leukemia associated nucleic acid or polypeptide in thebiological sample. As used herein, "contacting" means placing thebiological sample in sufficient proximity to the agent and under theappropriate conditions of, e.g., concentration, temperature, time, ionicstrength, to allow the specific interaction between the agent andleukemia associated nucleic acid or polypeptide that are present in thebiological sample. In general, the conditions for contacting the agentwith the biological sample are conditions known by those of ordinaryskill in the art to facilitate a specific interaction between a moleculeand its cognate (e.g., a protein and its receptor cognate, an antibodyand its protein antigen cognate, a nucleic acid and its complementarysequence cognate) in a biological sample. Exemplary conditions forfacilitating a specific interaction between a molecule and its cognateare described in U.S. Pat. No. 5,108,921, issued to Low et al.

The biological sample can be located in vivo or in vitro. For example,the biological sample can be a hematopoietic tissue in vivo and theagent specific for the leukemia associated nucleic acid or polypeptidecan be used to detect the presence of such molecules in thehematopoietic tissue (e.g., for imaging portions of the hematopoietictissue that express the leukemia associated gene products).Alternatively, the biological sample can be located in vitro (e.g., ablood sample, bone marrow biopsy, tissue extract). In a particularlypreferred embodiment, the biological sample can be a cell-containingsample, more preferably a sample containing hematopoietic cells.

Also a part of the invention are those nucleic acid sequences which alsocode for a preproTRH, tryptase-L or Oct-T1 polypeptide and whichhybridize under stringent conditions to a nucleic acid moleculeconsisting of the nucleotide sequence set forth in SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5 or SEQ ID NO:7, respectively. Such nucleic acids aretermed leukemia associated polypeptide precursors, and may be DNA, RNA,or composed of mixed deoxyribonucleotides and ribonucleotides. Theleukemia associated polypeptide precursors can also incorporatesynthetic non-natural nucleotides.

The invention thus encompasses other leukemia associated nucleic acids,some of which previously were identified in normal tissues. A leukemiaassociated nucleic acid or polypeptide is a nucleic acid or polypeptideexpressed preferentially in leukemias and solid forms of leukemia cellmalignancies, such as lymphomas. Various methods for determining theexpression of a nucleic acid and/or a polypeptide in normal and leukemiacells are known to those of skill in the art and are described furtherbelow. As used herein, leukemia associated polypeptides includeproteins, protein fragments, and peptides. In particular, leukemiaassociated polypeptides include TRAPs and TRAs.

The term "stringent conditions" as used herein refers to parameters withwhich the art is familiar. More specifically, stringent conditions, asused herein, refers to hybridization at 65° C. in hybridization buffer(3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% Bovine SerumAlbumin, 25 mM NaH₂ PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15Msodium chloride/0.15M sodium citrate, pH 7; SDS is sodium dodecylsulphate; and EDTA is ethylenediaminetetracetic acid. Afterhybridization, the membrane upon which the nucleic acid is transferredis washed at 2×SSC at room temperature and then at 0.1×SSC/0.1×SDS at65° C. SSC is 0.15M sodium chloride/0.15M sodium citrate, pH 7; SDS issodium dodecyl sulphate; and EDTA is ethylenediamine tetraacetic acid.

There are other conditions, reagents, and so forth which can be used,which result in the same degree of stringency (see, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York). The skilled artisan will be familiarwith such conditions, and thus they are not given here. It will beunderstood, however, that the skilled artisan will be able to manipulatethe conditions in a manner to permit the clear identification ofhomologs and alleles of tryptase-L nucleic acid molecules of theinvention. The skilled artisan also is familiar with the methodology forscreening cells, preferably cancer cells, and libraries for expressionof such molecules which then are routinely isolated, followed byisolation of the pertinent nucleic acid and sequencing.

In general homologs and alleles typically will share at least 40%nucleotide identity and/or at least 50% amino acid identity to thecoding region of leukemia associated nucleic acids, in some instanceswill share at least 50% nucleotide identity and/or at least 65% aminoacid identity and in still other instances will share at least 60%nucleotide identity and/or at least 75% amino acid identity.Watson-Crick complements of the foregoing nucleic acids also areembraced by the invention.

The nucleic acids disclosed herein are useful for determining theexpression of preproTRH, tryptase-L and Oct-T1 genes according tostandard hybridization procedures. The nucleic acids also can be used toexpress leukemia associated polypeptides in vitro or in vivo. Thenucleic acids also can be used to prepare fragments of such polypeptidesuseful for e.g., preparation of antibodies. Many other uses will beapparent to the skilled artisan.

In screening for related genes, such as tryptase-L family members, aSouthern blot may be performed using the foregoing conditions, togetherwith a radioactive probe. After washing the membrane to which thenucleic acid is finally transferred, the membrane can be placed againstx-ray film to detect the radioactive signal.

The invention also includes degenerate nucleic acids which includealternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue. Similarly, nucleotide sequencetriplets which encode other amino acid residues include, but are notlimited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT,AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons);AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucinecodons). Other amino acid residues may be encoded similarly by multiplenucleotide sequences. Thus, the invention embraces degenerate nucleicacids that differ from the biologically isolated nucleic acids in codonsequence due to the degeneracy of the genetic code.

The invention also provides isolated unique fragments of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or complements thereof. A uniquefragment is one that is a `signature` for the larger nucleic acid. Itis, for example, long enough to assure that its precise sequence is notfound in molecules outside of the tryptase-L family as defined herein.Unique fragments can be used as probes in Southern blot assays toidentify family members or can be used in amplification assays such asthose employing PCR. As known to those skilled in the art, large probessuch as 200 nucleotides or more are preferred for certain uses such asSouthern blots, while smaller fragments will be preferred for uses suchas PCR. Unique fragments also can be used to produce fusion proteins forgenerating antibodies or for generating immunoassay components. Uniquefragments further can be used as antisense molecules to inhibit theexpression of the leukemia associated nucleic acids and encoded proteinsof the invention, particularly for therapeutic purposes as described ingreater detail below.

As will be recognized by those skilled in the art, the size of theunique fragment will depend upon its conservancy in the genetic code.Thus, some regions of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7and complements thereof will require longer segments to be unique whileothers will require only short segments, typically between 12 and 32nucleotides (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 and 32 nucleotides long). Those skilled inthe art are well versed in methods for selecting such sequences,typically on the basis of the ability of the unique fragment toselectively distinguish the sequence of interest from non-familymembers. A comparison of the sequence of the fragment to those on knowndata bases typically is all that is necessary, although in vitroconfirmatory hybridization and sequencing analysis may be performed.

For any pair of PCR primers constructed and arranged to selectivelyamplify, for example, a tryptase-L nucleic acid, a tryptase-L specificprimer may be used. Such a primer is a contiguous stretch of tryptase-Lwhich hybridizes selectively to tryptase-L and not other tryptasenucleic acids. Such a specific primer would fully hybridize to acontiguous stretch of nucleotides only in tryptase-L, but wouldhybridize at most only in part to tryptase genes that do not share thenucleotides to which the tryptase-L specific primer binds. For efficientPCR priming and tryptase-L identification, the tryptase-L specificprimer should be constructed and arranged so it does not hybridizeefficiently at its 3' end to tryptase genes other than tryptase-L.Preferably the area of non-identity is at least one to four nucleotidesin length and forms the 3' end of the tryptase-L specific primer. Thekinetics of hybridization then will strongly favor hybridization at the5' end. In this instance, 3' initiated PCR extension will occur onlywhen both the 5' and 3' ends hybridize to the nucleic acid. Primers forselective amplification of tryptase-L preferably are selected fromportions of SEQ ID NO:3 which share lesser homology with tryptases otherthan tryptase-L. In such cases, selective amplification of tryptase-Lcan be achieved with one tryptase-L specific primer and one primer whichhybridizes to tryptases generically. Preferably, however, both primersare tryptase-L specific primers are described hereinabove. Exemplaryprimers include SEQ ID NO:11 and SEQ ID NO:12, which are derived fromSEQ ID NO:3, respectively. Other exemplary primers can differ from theabove by addition or deletion of 1, 2, 3, 4, 5, or more nucleotides fromthe 5' end of the primer.

Similarly, one of ordinary skill in the art can select primers from thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:7 for selectiveamplification of preproTRH or Oct-T1 mRNA sequences, respectively. Forexample, exemplary primers specific for preproTRH include SEQ ID NO:9and SEQ ID NO:10, which are derived from SEQ ID NO:1. Exemplary primersspecific for Oct-T1 include SEQ ID NO:13 and SEQ ID NO:14, which arederived from SEQ ID NO:7. For amplification of tryptase-L, primers canbe designed which are specific for each clone (i.e., which amplify aportion of either SEQ ID NO:3 or SEQ ID NO:5), or which amplify bothclones (i.e., SEQ ID NO:3 and SEQ ID NO:5). As demonstrated in theExamples below, primer pairs specific to preproTRH, tryptase-L or Oct-T1can be used to distinguish the expression of the genes in cells andtissues. Other exemplary primers can differ from the above by additionor deletion of 1, 2, 3, 4, 5, or more nucleotides from the 5' end of theprimers above. One of ordinary skill in the art can determine with nomore than routine experimentation the preferred primers for selectiveamplification of particular leukemia associated genes.

Additional methods which can distinguish nucleotide sequences ofsubstantial homology, such as ligase chain reaction ("LCR") and othermethods, will be apparent to skilled artisans.

As used herein with respect to nucleic acids, the term "isolated" means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5' and 3' restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

The invention also provides isolated polypeptides which include uniquefragments of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. Suchpolypeptides are useful, for example, alone or as fusion proteins togenerate antibodies, as a components of an immunoassay, or fordetermining the binding specificity of HLA molecules and/or CTL clonesfor preproTRH, tryptase-L and Oct-T1 proteins.

A unique fragment of a tryptase-L protein, for example, generally hasthe features and characteristics of unique fragments as discussed abovein connection with nucleic acids. As will be recognized by those skilledin the art, the size of the unique fragment will depend upon factorssuch as whether the fragment constitutes a portion of a conservedprotein domain. Thus, some regions of SEQ ID NO:4 (or SEQ ID NO:6), willrequire longer segments to be unique while others will require onlyshort segments, typically between 5 and 12 amino acids (e.g. 5, 6, 7, 8,9, 10, 11 and 12 amino acids long).

Unique fragments of a polypeptide preferably are those fragments whichretain a distinct functional capability of the polypeptide. Functionalcapabilities which can be retained in a unique fragment of a polypeptideinclude interaction with antibodies, interaction with other polypeptidesor fragments thereof, selective binding of nucleic acids, and enzymaticactivity. A tumor rejection antigen is an example of a unique fragmentof a tumor specific polypeptide which retains the functional capabilityof HLA binding and interaction with cytotoxic T lymphocytes. Tumorrejection antigens presented by HLA class I molecules typically are 9amino acids in length, although peptides of 8, 9 and 10 and more aminoacids also retain the capability to interact with HLA and cytotoxic Tlymphocyte to an extent effective to provoke a cytotoxic T lymphocyteresponse (see, e.g., Van den Eynde & Brichard, Curr. Opin. Immunol.7:674-681, 1995; Coulie et al., Stem Cells 13:393-403, 1995).

Those skilled in the art are well versed in methods for selecting uniqueamino acid sequences, typically on the basis of the ability of theunique fragment to selectively distinguish the sequence of interest fromnon-family members. A comparison of the sequence of the fragment tothose on known data bases typically is all that is necessary.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in tryptase-L polypeptides to providefunctionally active homologs of the foregoing polypeptides, i.e, thehomologs retain the functional capabilities of the preproTRH, tryptase-Lor Oct-T1 polypeptides. As used herein, a "conservative amino acidsubstitution" refers to an amino acid substitution which does not alterthe relative charge or size characteristics of the protein in which theamino acid substitution is made. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

Functionally equivalent variants of preproTRH, tryptase-L and/or Oct-T1polypeptides, i.e., variants of polypeptides which retain the functionof the natural polypeptides, can be prepared according to methods foraltering polypeptide sequence known to one of ordinary skill in the artsuch as are found in references which compile such methods, e.g.Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. For example, exemplaryfunctionally equivalent variants of the tryptase-L polypeptides includeconservative amino acid substitutions of SEQ ID NO:4 or and SEQ ID NO:6.Conservative amino-acid substitutions in the amino acid sequence oftryptase-L polypeptides to produce functionally equivalent variants oftryptase-L polypeptides typically are made by alteration of the nucleicacid encoding tryptase-L (SEQ ID NO:3, SEQ ID NO:5). Such substitutionscan be made by a variety of methods known to one of ordinary skill inthe art. For example, amino acid substitutions may be made byPCR-directed mutation, site-directed mutagenesis according to the methodof Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), orby chemical synthesis of a gene encoding a tryptase-L polypeptide. Whereamino acid substitutions are made to a small unique fragment of atryptase-L polypeptide, such as a 9 amino acid peptide, thesubstitutions can be made by directly synthesizing the peptide. Theactivity of functionally equivalent fragments of tryptase-L polypeptidescan be tested by cloning the gene encoding the altered tryptase-Lpolypeptide into a bacterial or mammalian expression vector, introducingthe vector into an appropriate host cell, expressing the alteredtryptase-L polypeptide, and testing for a functional capability of thetryptase-L polypeptides as disclosed herein. Functionally equivalentvariants of the preproTRH and Oct-T1 polypeptides can be prepared in alike manner.

As mentioned above, the invention embraces antisense oligonucleotidesthat selectively bind to a leukemia associated gene nucleic acidmolecule, including those encoding a preproTRH protein, a tryptase-Lprotein or a Oct-T1 protein, to decrease transcription and/ortranslation of leukemia associated genes. This is desirable in virtuallyany medical condition wherein a reduction in leukemia associated geneproduct expression is desirable, including to reduce any aspect of amalignant hemopathy cell phenotype attributable to leukemia associatedgene expression, such as expression of preproTRH, tryptase-L and/orOct-T1. Antisense molecules, in this manner, can be used to slow down orarrest such aspects of a malignant leukemia cell phenotype as found in,inter alia, leukemia and solid forms such as lymphoma.

As used herein, the term "antisense oligonucleotide" or "antisense"describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene. Those skilled in the art willrecognize that the exact length of the antisense oligonucleotide and itsdegree of complementarity with its target will depend upon the specifictarget selected, including the sequence of the target and the particularbases which comprise that sequence. It is preferred that the antisenseoligonucleotide be constructed and arranged so as to bind selectivelywith the target under physiological conditions, i.e., to hybridizesubstantially more to the target sequence than to any other sequence inthe target cell under physiological conditions. Based upon SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5 and/or SEQ ID NO:7, or upon allelic orhomologous genomic and/or DNA sequences, one of skill in the art caneasily choose and synthesize any of a number of appropriate antisensemolecules for use in accordance with the present invention. In order tobe sufficiently selective and potent for inhibition, such antisenseoligonucleotides should comprise at least 7 (Wagner et al., NatureBiotechnology 14:840-844, 1996) and, more preferably, at least 15consecutive bases which are complementary to the target. Mostpreferably, the antisense oligonucleotides comprise a complementarysequence of 20-30 bases. Although oligonucleotides may be chosen whichare antisense to any region of the gene or mRNA transcripts, inpreferred embodiments the antisense oligonucleotides correspond toN-terminal or 5' upstream sites such as translation initiation,transcription initiation or promoter sites. In addition, 3'-untranslatedregions may be targeted. Targeting to mRNA splicing sites has also beenused in the art but may be less preferred if alternative mRNA splicingoccurs. In addition, the antisense is targeted, preferably, to sites inwhich mRNA secondary structure is not expected (see, e.g., Sainio etal., Cell Mol. Neurobiol. 14(5):439-457, 1994) and at which proteins arenot expected to bind. Finally, although, SEQ ID NOs:1, 3, 5 and 7disclose cDNA sequences, one of ordinary skill in the art may easilyderive the genomic DNA corresponding to the cDNAs of SEQ ID NOs:1, 3, 5and 7. Thus, the present invention also provides for antisenseoligonucleotides which are complementary to the genomic DNAcorresponding to SEQ ID NOs:1, 3, 5 and 7. Similarly, antisense toallelic or homologous DNAs and genomic DNAs are enabled without undueexperimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of "natural" deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5' end of onenative nucleotide and the 3' end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include "modified" oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term "modified oligonucleotide" as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5' end of onenucleotide and the 3' end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic intemucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,is phosphate esters, alkylphosphonothioates, phosphoramidates,carbamates, carbonates, phosphate triesters, acetamidates, peptides, andcarboxymethyl esters.

The term "modified oligonucleotide" also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3' position and other than a phosphategroup at the 5' position. Thus modified oligonucleotides may include a2'-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. Modifiedoligonucleotides also can include base analogs such as C-5 propynemodified bases (Wagner et al., Nature Biotechnology 14:840-844, 1996).The present invention, thus, contemplates pharmaceutical preparationscontaining modified antisense molecules that are complementary to andhybridizable with, under physiological conditions, nucleic acidsencoding leukemia associated proteins, together with pharmaceuticallyacceptable carriers.

It will also be recognized from the examples that the invention embracesthe use of the preproTRH, tryptase-L and Oct-T1 sequences in expressionvectors, as well as to transfect host cells and cell lines, be theseprokaryotic (e.g., E. coli), or eukaryotic (e.g., CHO cells, COS cells,yeast expression systems and recombinant baculovirus expression ininsect cells). Especially useful are mammalian cells such as mouse,hamster, pig, goat, primate, etc. They can be of a wide variety oftissue types, including mast cells, fibroblasts, oocytes andlymphocytes, and they may be primary cells or cell lines. Specificexamples include dendritic cells, U293 cells, peripheral bloodleukocytes, bone marrow stem cells and embryonic stem cells. Theexpression vectors require that the pertinent sequence, i.e., thosenucleic acids described supra, be operably linked to a promoter. Ininstances in which a human HLA class I molecule presents tumor rejectionantigens derived from the preproTRH, tryptase-L and Oct-T1 genes, theexpression vector may also include a nucleic acid sequence coding forthe HLA molecule that presents any particular tumor rejection antigenderived from these genes and polypeptides. Alternatively, the nucleicacid sequence coding for such a HLA molecule can be contained within aseparate expression vector. In a situation where the vector containsboth coding sequences, the single vector can be used to transfect a cellwhich does not normally express either one. Where the coding sequencesfor the tumor rejection antigen precursor and the HLA molecule whichpresents it are contained on separate expression vectors, the expressionvectors can be cotransfected. The tumor rejection antigen precursorcoding sequence may be used alone, when, e.g. the host cell alreadyexpresses a HLA molecule which presents a TRA derived from preproTRH,tryptase-L and/or Oct-T1 TRAPs. Of course, there is no limit on theparticular host cell which can be used. As the vectors which contain thetwo coding sequences may be used in any antigen-presenting cells ifdesired, and the gene for tumor rejection antigen precursor can be usedin host cells which do not express a HLA molecule which presents apreproTRH, tryptase-L and/or Oct-T1 TRA. Further, cell-freetranscription systems may be used in lieu of cells.

As used herein, a "vector" may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmidsand phagemids. A cloning vector is one which is able to replicate in ahost cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g. β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques. Preferred vectors are those capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe "operably" joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5' regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5' non-transcribing and 5' non-translatingsequences involved with the initiation of transcription and translationrespectively, such as a TAT box, capping sequence, CAAT sequence, andthe like. Especially, such 5' non-transcribing regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5' leader or signal sequences, 5' or 3'. The choice and designof an appropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. SeeMolecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. Cells are genetically engineered by the introduction intothe cells of heterologous DNA (RNA) encoding the preproTRH, tryptase-Lor Oct-T1 tumor specific polypeptide or fragment or variant thereof.That heterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen), which contains an Epstein Barr virus (EBV) originof replication, facilitating the maintenance of plasmid as a multicopyextrachromosomal element. Another expression vector is the pEF-BOSplasmid containing the promoter of polypeptide Elongation Factor 1α,which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992). The use of the adenovirus as an Adeno.P1A recombinant isdisclosed by Warnier et al., in intradermal injection in mice forimmunization against P1A (Int. J. Cancer. 67:303-310, 1996).

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

The invention also involves agents which bind to leukemia associatedpolypeptides including preproTRH, tryptase-L and Oct-T1, and in certainembodiments preferably to unique fragments of the preproTRH, tryptase-Land Oct-T1 polypeptides. Such binding partners can be used in screeningassays to detect the presence or absence of a preproTRH, tryptase-L orOct-T1 polypeptide and in purification protocols to isolate preproTRH,tryptase-L or Oct-T1 polypeptides. Likewise, such binding partners canbe used to selectively target drugs, toxins or other molecules toleukemia cells which present preproTRH, tryptase-L or Oct-T1 leukemiaassociated polypeptides. In this manner, cells present in solid ornon-solid tumors which express preproTRH, tryptase-L or Oct-T1 leukemiaassociated polypeptides can be treated with cytotoxic compounds.

The invention, therefore, involves antibodies or fragments of antibodieshaving the ability to selectively bind to preproTRH, tryptase-L orOct-T1 leukemia associated polypeptides, and preferably to uniquefragments thereof. Antibodies include polyclonal and monoclonalantibodies, prepared according to conventional methodology.

The antibodies of the present invention thus are prepared by any of avariety of methods, including administering protein, fragments ofprotein, cells expressing the protein or fragments thereof and the liketo an animal to induce polyclonal antibodies. The production ofmonoclonal antibodies is according to techniques well known in the art.As detailed herein, such antibodies may be used for example to identifytissues expressing protein or to purify protein. Antibodies also may becoupled to specific labeling agents for imaging or to antitumor agents,including, but not limited to, methotrexate, radioiodinated compounds,toxins such as ricin, other cytostatic or cytolytic drugs, and so forth.Antibodies prepared according to the invention also preferably arespecific for the TRA/HLA complexes described herein.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc' and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc' region has been enzymaticallycleaved, or which has been produced without the pFc' region, designatedan F(ab')₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of "humanized" antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc' regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as "chimeric" antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab')₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab')₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies. Thus, the invention involves polypeptides ofnumerous size and type that bind specifically to leukemia associatedpolypeptides including preproTRH, tryptase-L or Oct-T1. Thesepolypeptides may be derived also from sources other than antibodytechnology. For example, such polypeptide binding agents can be providedby degenerate peptide libraries which can be readily prepared insolution, in immobilized form or as phage display libraries.Combinatorial libraries also can be synthesized of peptides containingone or more amino acids. Libraries further can be synthesized ofpeptoids and non-peptide synthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent a completely degenerate or biased array. Onethen can select phage-bearing inserts which bind to a preproTRH,tryptase-L or Oct-T1 leukemia associated polypeptide. This process canbe repeated through several cycles of reselection of phage that bind toa preproTRH, tryptase-L or Oct-T1 polypeptide. Repeated rounds lead toenrichment of phage bearing particular sequences. DNA sequence analysiscan be conducted to identify the sequences of the expressedpolypeptides. The minimal linear portion of the sequence that binds tothe preproTRH, tryptase-L or Oct-T1 polypeptide can be determined. Onecan repeat the procedure using a biased library containing insertscontaining part or all of the minimal linear portion plus one or moreadditional degenerate residues upstream or downstream thereof. Thus, theleukemia associated polypeptides of the invention can be used to screenpeptide libraries, including phage display libraries, to identify andselect peptide binding partners of the leukemia associated polypeptidesof the invention. Such molecules can be used, as described, forscreening assays, for diagnostic assays, for purification protocols orfor targeting drugs, toxins and/or labeling agents (e.g. radioisotopes,fluorescent molecules, etc.) to cells which express leukemia associatedgenes such as those leukemia cells which present preproTRH, tryptase-Lor Oct-T1 polypeptides on the cell surface. Such binding agent moleculescan also be prepared to bind complexes of an preproTRH, tryptase-L orOct-T1 polypeptide and an HLA molecule by selecting the binding agentusing such complexes. Drug molecules that would disable or destroyleukemia cells which express such complexes or preproTRH, tryptase-L orOct-T1 polypeptides are known to those skilled in the art and arecommercially available. For example, the immunotoxin art providesexamples of toxins which are effective when delivered to a cell by anantibody or fragment thereof. Examples of toxins includeribosome-damaging toxins derived from plants or bacterial such as ricin,abrin, saporin, Pseudomonas endotoxin, diphtheria toxin, A chain toxins,blocked ricin, etc.

The skilled artisan can determine which HLA molecule binds to tumorrejection antigens derived from preproTRH, tryptase-L and/or Oct-T1tumor rejection antigen precursors by, e.g., experiments utilizingantibodies to block specifically individual HLA class I molecules. Forexample, antibodies which bind selectively to HLA-A2 will preventefficient presentation of TRAs specifically presented by HLA-A2. Thus,if TRAs derived from leukemia associated genes such as preproTRH,tryptase-L and/or Oct-T1 are presented by HLA-A2, then the inclusion ofanti-HLA-A2 antibodies in an in vitro assay will block the presentationof these TRAs. An assay for determining the nature of the HLA moleculeis found in U.S. patent application Ser. No. 08/530,569. Briefly, indetermining the HLA molecule type, inhibition experiments were carriedout where the production of tumor necrosis factor (TNF) by cytotoxic Tlymphocyte (CTL) clone 263/17 was tested in the presence of monoclonalantibodies directed against HLA molecules or against CD4/CD8 accessorymolecules. Four monoclonal antibodies were found to inhibit theproduction of TNF by CTL 263/17: monoclonal antibody W6/32, which isdirected against all HLA class I molecules (Parham et al., J. Immunol.123:342, 1979), antibody B1.23.2 which recognizes HLA-B and C molecules(Rebai et al., Tissue Antigens 22:107, 1983), antibody ME-1 whichspecifically recognizes HLA-B7 (Ellis et al., Hum. Immunol. 5:49, 1982)and antibody B9.4.1 against CD8. No inhibition was found with antibodiesdirected against HLA Class II DR molecules (L243: Lampson et al., J.Immunol. 125:293, 1980), against HLA-A3 (GAPA 3: Berger et al.,Hybridoma 1:87, 1982) or against CD4 (13B.8.82). The conclusion was thatCTL 263/17 was of the CD8 type, and recognized an antigen presented byHLA-B7. Similar experiments using widely available anti-HLA antibodiescan be performed to determine the nature of a HLA molecule.

The invention as described herein has a number of uses, some of whichare described herein. First, the invention permits the artisan todiagnose a disorder characterized by expression of the TRAP. Thesemethods involve determining expression of the TRAP gene, and/or TRAsderived therefrom, such as a TRA presented by HLA-A2, HLA-A26, HLA-B7,etc. In the former situation, such determinations can be carried out viaany standard nucleic acid determination assay, including the polymerasechain reaction, or assaying with labeled hybridization probes. In thelatter situation, assaying with binding partners for complexes of TRAand HLA, such as antibodies, is especially preferred. An alternatemethod for determination is a TNF release assay, of the type describedsupra.

The isolation of the TRAP gene also makes it possible to isolate theTRAP molecule tself, especially TRAP molecules containing the acmcninoacid sequences coded for by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQID NO:8. Other TRAPs or TRAs encoded by leukemia associated genes andrecognized by other CTL clones and/or presented by other HLA moleculesmay be isolated by the procedures detailed herein. (There are numerousHLA molecules known to those skilled in the art, including but notlimited to, those encoded by HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-Ggenes.) A variety of methodologies well-known to the skilledpractitioner can be utilized to obtain isolated TRAP molecules. Theprotein may be purified from cells which naturally produce the protein.Alternatively, an expression vector may be introduced into cells tocause production of the protein. In another method, mRNA transcripts maybe microinjected or otherwise introduced into cells to cause productionof the encoded protein. Translation of mRNA in cell-free extracts suchas the reticulocyte lysate system also may be used to produce protein.Peptides comprising TRAs of the invention may also be synthesized invitro. Those skilled in the art also can readily follow known methodsfor isolating proteins in order to obtain isolated TRAPs and/or TRAsderived therefrom. These include, but are not limited to,immunochromotography, HPLC, size-exclusion chromatography, ion-exchangechromatography and immune-affinity chromatography.

These isolated molecules when processed and presented as the TRA, or ascomplexes of TRA and HLA, such as HLA-A2, HLA-A26 or HLA-B7, etc. may becombined with materials such as adjuvants to produce vaccines useful intreating disorders characterized by expression of the TRAP molecule. Inaddition, vaccines can be prepared from cells which present the TRA/HLAcomplexes on their surface, such as non-proliferative cancer cells,non-proliferative transfectants, etcetera. In all cases where cells areused as a vaccine, these can be cells transfected with coding sequencesfor one or both of the components necessary to provoke a CTL response,or be cells which already express both molecules without the need fortransfection. Vaccines also encompass naked DNA or RNA, encoding aleukemia associated TRA or precursor thereof, which may be produced invitro and administered via injection, particle bombardment, nasalaspiration and other methods. Vaccines of the "naked nucleic acid" typehave been demonstrated to provoke an immunological response includinggeneration of CTLs specific for the peptide encoded by the naked nucleicacid (Science 259:1745-1748, 1993). When "disorder" is used herein, itrefers to any pathological condition where the tumor rejection antigenprecursor is expressed. An example of such a disorder is cancer,leukemias and lymphomas in particular.

In addition, vaccines can be prepared from cells which present theTRA/HLA complexes on their surface, such as non-proliferative cancercells, non-proliferative transfectants, etcetera. In all cases wherecells are used as a vaccine, these can be cells transfected with codingsequences for one or both of the components necessary to provoke a CTLresponse, or be cells which already express both molecules without theneed for transfection.

Therapeutic approaches based upon the disclosure are premised on aresponse by a subject's immune system, leading to lysis of TRApresenting cells, such as HLA-B7 cells. One such approach is theadministration of autologous CTLs specific to the complex to a subjectwith abnormal cells of the phenotype at issue. It is within the skill ofthe artisan to develop such CTLs in vitro. Generally, a sample of cellstaken from a subject, such as blood cells, are contacted with a cellpresenting the complex and capable of provoking CTLs to proliferate. Thetarget cell can be a transfectant, such as a COS cell of the typedescribed supra. These transfectants present the desired complex oftheir surface and, when combined with a CTL of interest, stimulate itsproliferation. COS cells, such as those used herein are widelyavailable, as are other suitable host cells. Specific production of aCTL is well known to one of ordinary skill in the art. The clonallyexpanded autologous CTLs then are administered to the subject. OtherCTLs specific to preproTRH, tryptase-L and/or Oct-T1 may be isolated andadministered by similar methods.

To detail a therapeutic methodology, referred to as adoptive transfer(Greenberg, J. Immunol. 136(5): 1917, 1986; Riddel et al., Science 257:238, 1992; Lynch et al, Eur. J. Immunol. 21: 1403-1410, 1991; Kast etal., Cell 59: 603-614, 1989), cells presenting the desired complex arecombined with CTLs leading to proliferation of the CTLs specificthereto. The proliferated CTLs are then administered to a subject with acellular abnormality which is characterized by certain of the abnormalcells presenting the particular complex. The CTLs then lyse the abnormalcells, thereby achieving the desired therapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/TRA complex. This can bedetermined very easily, as the art is very familiar with methods foridentifying cells which present a particular HLA molecule, as well ashow to identify cells expressing DNA of the pertinent sequences, in thiscase a leukemia associated gene sequence. Once cells presenting therelevant complex are identified via the foregoing screening methodology,they can be combined with a sample from a patient, where the samplecontains CTLs. If the complex presenting cells are lysed by the mixedCTL sample, then it can be assumed that a leukemia associated genederived TRA is being presented, and the subject is an appropriatecandidate for the therapeutic approaches set forth supra.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach is the use of non-proliferativecells expressing the complex. The cells used in this approach may bethose that normally express the complex, such as irradiated tumor cellsor cells transfected with one or both of the genes necessary forpresentation of the complex. Chen et al., Proc. Natl. Acad. Sci. USA 88:110-114 (1991) exemplifies this approach, showing the use of transfectedcells expressing HPV E7 peptides in a therapeutic regime. Various celltypes may be used. Similarly, vectors carrying one or both of the genesof interest may be used. Viral or bacterial vectors are especiallypreferred. For example, nucleic acids which encode a preproTRH,tryptase-L or Oct-T1 TRA may be operably linked to promoter and enhancersequences which direct expression of the preproTRH, tryptase-L or Oct-T1TRA in certain tissues or cell types. The nucleic acid may beincorporated into an expression vector. Expression vectors may beunmodified extrachromosomal nucleic acids, plasmids or viral genomesconstructed or modified to enable insertion of exogenous nucleic acids,such as those encoding preproTRH, tryptase-L or Oct-T1 TRAs. Nucleicacids encoding a preproTRH, tryptase-L or Oct-T1 TRA also may beinserted into a retroviral genome, thereby facilitating integration ofthe nucleic acid into the genome of the target tissue or cell type. Inthese systems, the gene of interest is carried by a microorganism, e.g.,a Vaccinia virus, retrovirus or the bacteria BCG, and the materialsdefacto "infect" host cells. The cells which result present the complexof interest, and are recognized by autologous CTLs, which thenproliferate.

A similar effect can be achieved by combining a TRAP or a stimulatoryfragment thereof with an adjuvant to facilitate incorporation into HLApresenting cells in vivo. The TRAP is processed to yield the peptidepartner of the HLA molecule while the TRA is presented without the needfor further processing. Generally, subjects can receive an intradermalinjection of an effective amount of a preproTRH, tryptase-L and/orOct-T1 encoded TRAP, and/or TRAs derived therefrom. Initial doses can befollowed by booster doses, following immunization protocols standard inthe art.

As part of the immunization protocols, substances which potentiate theimmune response may be administered with nucleic acid or peptidecomponents of a cancer vaccine. Such immune response potentiatingcompound may be classified as either adjuvants or cytokines. Adjuvantsmay enhance the immunological response by providing a reservoir ofantigen (extracellularly or within macrophages), activating macrophagesand stimulating specific sets of lymphocytes. Adjuvants of many kindsare well known in the art; specific examples include MPL (SmithKlineBeecham), a congener obtained after purification and acid hydrolysis ofSalmonella Minnesota Re 595 lipopolysaccharide, QS21 (SmithKlineBeecham), a pure QA-21 saponin purified from Quillja saponaria extract,and various water-in-oil emulsions prepared from biodegradable oils suchas squalene and/or tocopherol. Cytokines are also useful in vaccinationprotocols as a result of lymphocyte stimulatory properties. Manycytokines useful for such purposes will be known to one of ordinaryskill in the art, including interleukin-12 (IL-12) which has been shownto enhance the protective effects of vaccines (Science 268: 1432-1434,1995).

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The term "pharmaceutically acceptable" means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredients. The term "physiologically acceptable" refers toa non-toxic material that is compatible with a biological system such asa cell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. When antibodies are used therapeutically, a preferred routeof administration is by pulmonary aerosol. Techniques for preparingaerosol delivery systems containing antibodies are well known to thoseof skill in the art. Generally, such systems should utilize componentswhich will not significantly impair the biological properties of theantibodies, such as the paratope binding capacity (see, for example,Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences,18th edition, 1990, pp 1694-1712). Those of skill in the art can readilydetermine the various parameters and conditions for producing antibodyaerosols without resort to undue experimentation. When using antisensepreparations of the invention, slow intravenous administration ispreferred.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The invention also contemplates gene therapy. The procedure forperforming ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346and in exhibits submitted in the file history of that patent, all ofwhich are publicly available documents. In general, it involvesintroduction in vitro of a functional copy of a gene into a cell(s) of asubject which contains a defective copy of the gene, and returning thegenetically engineered cell(s) to the subject. The functional copy ofthe gene is under operable control of regulatory elements which permitexpression of the gene in the genetically engineered cell(s). Numeroustransfection and transduction techniques as well as appropriateexpression vectors are well known to those of ordinary skill in the art,some of which are described in PCT application WO95/00654. In vivo genetherapy using vectors such as adenovirus also is contemplated accordingto the invention.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a pharmaceutical preparation thatalone, or together with further doses, stimulates the desired response.In the case of treating cancer, the desired response is inhibiting theprogression of the cancer. This may involve only slowing the progressionof the disease temporarily, although more preferably, it involveshalting the progression of the disease permanently. This can bemonitored by routine methods or can be monitored according to diagnosticmethods of the invention discussed herein.

Where it is desired to stimulate an immune response using a therapeuticcomposition of the invention, this may involve the stimulation of ahumoral antibody response resulting in an increase in antibody titer inserum, a clonal expansion of cytotoxic lymphocytes, or some otherdesirable immunologic response. It is believed that doses of immunogensranging from one nanogram/kilogram to 100 milligrams/kilogram, dependingupon the mode of administration, would be effective. The preferred rangeis believed to be between 500 nanograms and 500 micrograms per kilogram.The absolute amount will depend upon a variety of factors, including thematerial selected for administration, whether the administration is insingle or multiple doses, and individual patient parameters includingage, physical condition, size, weight, and the stage of the disease.These factors are well known to those of ordinary skill in the art andcan be addressed with no more than routine experimentation.

EXAMPLES Example 1 Representational Difference Analysis--SubtractivecDNA hybridizations between leukemia and normal leukoccytes.

1. Procedure

Tester population: PBL from a female patient (SIAX, LB-1079) with newlydiagnosed acute myelogenous leukemia (FAB-M2 subtype), with achromosomal rearrangement t(8;21)(q22;q22) detected by cytogeneticanalysis. The PBL were collected by leukapheresis before startingchemotherapy (approximately 70% of the PBL were leukemia blast cells),purified on a Lymphoprep (Ficol1) gradient, washed and frozen in culturemedium and DMSO at -80° C. For RNA preparation, 6×10⁸ cells were thawed,washed and centrifuged as a dry cell pellet, containing 4.6×10⁸ cells.

Driver population: PBL from a normal person were collected byleukapheresis, they were purified on a Lymphoprep (Ficol1) gradient,washed and frozen at -80° C. as a dry cell pellet after centrifugation,containing 5×10⁸ cells.

Total RNA preparation: The guanidinium isothiocyanate/cesium chlorideprocedure was used L. G. Davis, M. D. Dibner, J. F. Battey, BasicMethods in Molecular Biology, Elsevier, N.Y., 1986, p. 130-135!. 390 μgand 50 μg were obtained from tester and driver populations respectively.

mRNA purification: Oligo-dT columns (Pharmacia mRNA Purification Kit,Pharmacia, Uppsala, Sweden) were used according to the manufacturer'sprotocol.

cDNA preparation: cDNA was prepared according to the manufacturer'sprotocol (Amersham, cDNA Synthesis Module). 6 μg and 2 μg were obtainedfrom tester and driver populations respectively.

Difference analysis: The cDNA Difference Analysis Protocol of Hubank andSchatz (Nucleic Acids Res. 22: 5640-5648, 1994) was followed for thepreparation of tester representation (TR), driver representation (DR),difference product 1 and difference product 2. Some particularitiesconcerning the preparation of tester representation must bementioned: 1) only 600 ng (and not 1.2 μg have been used in the initialligation of adaptors; 2) the PCR reaction has been divided in 40aliquots. Some particularities concerning the preparation of differenceproduct 1 (DP1) must be mentioned: only 0.2 μg TR and 20 μg DR (and not0.4 and 40 μg) were used in 5 μl hybridization buffer. Someparticularities concerning the preparation of difference product 2 (DP2)must be mentioned: only 2.5 ng TR and 20 μg DR (and not 50 ng and 40 μg)were used in 5 μl hybridization buffer. Finally, no third differenceproduct could be obtained.

Cloning of the tester representation and difference products 1 and 2:The cDNAs of the tester representation and difference products 1 and 2obtained after digestion with DpnII were cloned by common ligation intothe BamHI cloning site of vector pTZ18R, digested with BamHI (DpnII andBamHI cohesive ends are mutually compatible) and dephosphorylated withCalf Intestinal Phosphatase. The ligation products were used totransform competent Top10F' bacteria by electroporation. Transformedbacteria were selected on agar plates with ampicillin, their plasmid DNAwas purified by miniprep DNA extraction (Qiaprep, Qiagen), and analyzedby BamHI digestion and DNA sequencing.

2. Results

Sequencing of 16 clones with DP2 products: Some clones contained morethan 1 insert. Note also that 1 single mRNA can provide more than oneamplified DpnII restriction fragment. Because two thirds of theindividual inserts were derived from the myeloperoxidase (MPO) mRNA,more bacterial colonies were isolated after hybridization with amyeloperoxydase-specific oligonucleotide probe. 48 MPO-negative colonieswere selected and grown with ampicillin, their plasmid DNA was purifiedby miniprep DNA extraction (Qiaprep, Qiagen), and analysed by DNAsequencing.

Conclusions: The majority of cDNA fragments amplified by the RDA method,using leukemia cells as tester population, and normal PBL as driverpopulation, derived from the myeloperoxidase mRNA. The MPO gene ishighly expressed in normal and leukemic myeloid progenitor cells, butnot in differentiated white blood cells. This is the reason why derivedcDNA fragments are amplified by RDA. This also explains the presence ofCD34 which was also amplified by RDA when normal PBL are used as thedriver population. The presence of cDNA fragments derived from highlyexpressed ubiquitous RNAs (28S and 40S ribosomal RNA genes) probablyresults from a residual background after two cycles of differentialhybridization. Some cDNA fragments are derived from known genes.

For the last two categories, we have designed specific PCR primer pairs,derived from the sequence obtained from the cloned fragments. RT-PCR wasperformed on a few leukemia, normal PBL and normal bone marrow samples.The genes expressed only in leukemia samples were further tested fortheir expression on a larger panel of normal and leukemic tissues. Onlythree genes appear to show a leukemia-specific expression pattern:

The prepro TRH gene.

The gene encoding an mRNA with strong (but not complete) homology withthe five known tryptase genes. This gene will be further referred to astryptase-L (L for leukemia).

An apparently new gene, further referred to as SIAX DP2-64.

The cDNAs of these three genes have been cloned from a cDNA libraryobtained from the same leukemia (SIAX, LB-1079), and sequenced. We havetested their expression by RT-PCR on normal tissue, leukemia and solidtumor samples. These data are further detailed in the following pages.

Example 2 Pre-pro-thyrotropin-releasing hormone gene (preproTRH)

Cloning the cDNA: The cDNA library NVB32 was prepared as follows: TotalRNA was extracted from the thawed leukemic PBL obtained from patientSIAX, LB-1079 (see tester population, RDA protocol, Example 1). Poly-ARNA was purified on oligo-dT columns (Pharmacia mRNA Purification Kit),according to the manufacturer's protocol. cDNA was prepared withSuperscript kit (Gibco-BRL), according to the manufacturer's protocol,with random primers. The cDNA fragments were ligated to the EcoRIadaptors, fractionated on a chromatography column (4 fractions: A, B, C,and D were obtained) and ligated into the EcoRI digested anddephosphorylated pcDSRalpha vector. The ligation products were used totransform Top10F' competent bacteria by electroporation. Transformedbacteria colonies were obtained after selective growth with ampicillin,pooled with respect to fraction, and kept frozen at -80° C. Fraction Bappeared to have the largest inserts.

The B fraction of the NVB329 cDNA library was screened for preproTRHclones by hybridizing 12,000 colonies with a ³² P-labelled PCR probeamplified from SIAX cDNA. Sixteen colonies were found to be positive,with two of them containing a plasmid with a ˜1.6 kb insert,corresponding to the predicted mRNA size. Only one of both recombinantplasmids (clone CHM327-3A/8) had its insert oriented in the rightdirection.

2. Sequencing: Clone CHM327-3A/8 has been fully sequenced (SEQ ID NO:1;see FIG. 1), using the Delta-Taq Sequencing Kit (Amersham). Its sequenceis completely identical to the published sequence of preproTRH, exceptfor the presence of an additional guanosine in the leukemia cDNA, in the3' untranslated region (the protein sequence is not affected by thisdifference).

3. Expression of the gene: Expression of the preproTRH gene in normal,leukemia and solid tumor tissues was tested by RT-PCR, as detailed inthe protocol herein.

a. expression in normal tissues: A positive signal was found in a fetalbrain sample. Absence of PCR amplification was found in the followingsamples: adult brain, colon, liver, ovary, skin, placenta, lung, kidney,testis, endometrium, bladder, normal peripheral blood leukocytes, normalbone marrow. These results are similar to the expression pattern foundin the literature, where the preproTRH gene is found expressed inhypothalamus and other parts of the central nervous system.

b. expression in solid tumors: The following tumor tissues were tested,and were found to be negative for the expression of the preproTRH gene:malignant melanoma, breast cancer, laryngeal carcinoma, lung NSCLC,bladder carcinoma, stomach cancer, lung SCLC, testicle tumor, uterinecarcinoma, renal carcinoma, colon carcinoma, tongue cancer, esophagealcancer, ovarian cancer, sarcoma, skin carcinoma. Note thatimmunoreactive TRH has been detected in human tumors, derived from theneural crest (Wilber, J. F., Clin. Endocr. 59: 3, 1984).

c. Expression in malignant hemopathies: the summary

acute myeloid leukemias: 11 positive samples (49 tested)

chronic myeloid leukemias: no positive sample (5 tested)

acute lymphoid leukemias: 4 positive samples (15 tested)

chronic lymphoid leukemias: no positive sample (2 tested)

multiple myeloma: no positive sample (1 tested)

Thus, the expression of the preproTRH gene is found in 23% of acuteleukemia samples.

The results can also be presented in relation with the most frequentchromosomal abnormalities found in the acute leukemias:

t(9;22)(q34;q11): 1 positive sample (3 tested)

t(8;21)(q22;q22): 11 positive samples (12 tested)

t(3;21)(q26;q22): 1 positive sample (2 tested)

t(12;21)(p13;q22): 2 positive samples (2 tested)

Inv(16)(p13;q22): 2 positive samples (8 tested)

t(15;17)(q22;q21): no positive samples (4 tested)

11q23 rearrangement: no positive samples (3 tested)

trisomy 8: no positive samples (4 tested)

del 5/5q or del 7/7q: 3 positive samples (8 tested)

There is a clear correlation between the preproTRH gene expression andthe acute leukemias with rearrangement of the AML1 gene, located on21q22, and encoding the AMLcl transcription factor.

4. Conclusion: The preproTRH gene is expressed in human acute leukemiacells. This is a new concept. The expression of the gene is related tochromosomal rearrangements involving the AML1 gene, which are involvedin leukemogenesis.

This gene is expressed in normal tissues located in the central nervoussystem. However, the preproTRH protein is processed through specializedenzymatic pathways in neuron hypothalamic cells to produce the modifiedtripeptide neurohormone TRH. PreproTRH may be processed differently inleukemic cells. It could accumulate in cell compartments where peptidesare efficiently processed and presented to HLA class I molecules. Inthis case, leukemic cells would carry strong antigenic peptides, whileneurons would not. Moreover, cells from the CNS are protected againstthe cell-mediated immunune system.

The preproTRH gene is expressed in leukemic cells, but not in normalbone marrow, nor in normal PBL. Therefore, its specific and sensitivedetection by RT-PCR or other methods is potentially useful as aleukemia-specific tumor marker, for the detection of minimal residualdisease, or for the quantitative evaluation of response to treatmentafter induction chemotherapy.

Example 3 Tryptase-L

1. Cloning the cDNA: The cDNA library NVB329 was prepared as detailed inExample 2 above. The B fraction of the NVB329 cDNA library was screenedfor tryptase-L clones by hybridizing 40,000 colonies with a ³³P-labelled oligonucleotide probe derived from the sequence of theamplified fragment SIAX DP2-04. Three colonies were found to bepositive, and each contained a plasmid (NVB352/1, 2, 3) with a ±2.2-2.5kb insert oriented in the right direction.

2. Sequencing: Two of the three independent inserts NVB352/1 andNVB352/3 have been sequenced and the partial sequence of clone NVB352/2has been determined (NVB352/1=SEQ ID NO:3; NVB352/3=SEQ ID NO:5; seeFIGS. 2 and 3), using the Delta-Taq Sequencing Kit (Amersham). The threecDNAs come from the same primary RNA transcript, whose sequence has astrong homology with the sequence of the five published tryptase mRNAs(tryptases alpha, beta, I, II, III). The three cDNA clones contain oneor two segments that are not present in the tryptase mRNA sequence, andthat contain consensus nucleotides similar to those found in thebeginning and at the end of introns. The genomic DNAs of the five knowntryptase genes contain introns at the same locations. Therefore, weconsider that these additional sequences in the 3 cDNA clones areunspliced introns. The deduced protein sequence, if we exclude theunspliced intron sequences, is very similar to the tryptase proteins,and the consensus amino acids for the serine protease activity areconserved. Note that the protein sequence is modified by the unsplicedintrons in the mRNA.

3. Expression of the gene: Expression of the tryptase-L gene in normal,leukemia and solid tumor tissues was tested by RT-PCR, as detailed inthe protocol herewith.

a. Expression in normal tissues: No positive signal was found in thefollowing normal tissue samples: fetal and adult brain, colon, liver,ovary, skin, placenta, lung, kidney, testis, endometrium, bladder,normal peripheral blood leukocytes, normal bone marrow. Note thattryptases are genes specifically expressed in mastocytes. We have notyet tested tryptase-L expression in normal mastocytes by RT-PCR.

b. Expression in solid tumors: The following tumor tissues were tested,and were found to be negative for the expression of the tryptase-L gene:malignant melanoma, breast cancer, laryngeal carcinoma, lung NSCLC,bladder carcinoma, stomach cancer, lung SCLC, testicle tumor, uterinecarcinoma, renal carcinoma, colon carcinoma, tongue cancer, esophagealcancer, varian cancer, sarcoma, skin carcinoma.

c. Expression in malignant hemopathies: Thirteen samples have beentested to date (detailed data are enclosed herewith). Six are positivefor the expression of tryptase-L, all of them are AMLs. Due to the smallsample size, we cannot draw any statistical conclusions, but it isnoteworthy that three of the positive samples are AMLs with a t(8;21)translocation.

4. Conclusion: The tryptase-L gene is expressed in human acute myeloidleukemia cells. Its expression has not been found in the normal tissuestested, but it is possible that normal mastocytes express the gene. Theexpression of the gene is possibly related to the t(8;21) chromosomalrearrangements.

The tryptase-L gene is expressed in leukemic cells, but not in normalbone marrow, nor in normal PBL. Therefore, its specific and sensitivedetection by RT-PCR and other methods is useful as a leukemia-specifictumor marker, for the detection of minimal residual disease, or for thequantitative evaluation of response to treatment after inductionchemotherapy.

Example 4 SIAX DP2-64/Oct-T1

1. Cloning the eDNA: The cDNA library NVB329 was prepared as detailed inExample 2. The B fraction of the NVB329 cDNA library was screened forSIAX DP2-64 clones by hybridizing 40,000 colonies with a ³² P-labelledPCR probe amplified from SIAX cDNA. One colony was found to be positive(clone CHM329/2-15). The same library was rescreened for SIAX DP2-64clones by hybridizing 40,000 colonies with a ³² P-labelled PCR probeamplified from SIAX cDNA. 11 colonies were found to be positive (clonesCHM363/3, 5, 8).

2. Sequencing: Clone CHM329/2-15 has been fully sequenced (SEQ ID NO:7;see FIGS. 4-6), using the Delta-Taq Sequencing Kit (Amersham). Its 3'half has no homology with known genes, while its 5' is completelyidentical to the 3' end of the Oct-T1 gene, a member of the POU familyof transcription factors. (Bhargava A. K. et al., Differentialexpression of 4 members of the POU family of proteins in activated andPMA-treated Jurkat T cells. Proc.Natl.Acad.Sci. 90:10260-10264, November1993).

Clones CHM363/3, CHM363/5 and CHM363/8 were sequenced in their 5'extremities, each of which were completely homologous to the Oct-T1 mRNAsequence (see FIG. 4). Clone CHM363/5 has been almost entirely sequenced(see FIG. 5), and is almost completely identical to the Oct-T1 sequence,with minor differences in the 3' untranslated region. Therefore, we canconclude that our SIAX DP2-64 is identical to the previously identifiedOct-T1 mRNA. It appears that the published sequence lacks the 3'extremity. We have cloned and sequenced the missing 600 base pairstogether with the poly-A tail.

3. Expression of the gene: Expression of the Oct-T1 gene in normal,leukemia and solid tumor tissues was tested by RT-PCR, as detailed inthe protocol herewith.

a. Expression in normal tissue: No positive signal was found in thefollowing samples: adult brain, colon, liver, ovary, skin, placenta,lung, kidney, endometrium, bladder, normal peripheral blood leukocytes,normal bone marrow. A positive signal was detected in all the testissamples tested, with the exception of a fetal testis sample.

b. Expression in solid tumors: The following tumor tissues were tested,and were found to be negative for the expression of the Oct-T1 gene:malignant melanoma, breast cancer, laryngeal carcinoma, lung NSCLC,bladder carcinoma, stomach cancer, lung SCLC, testicle tumor, uterinecarcinoma, renal carcinoma, colon carcinoma, tongue cancer, esophagealcancer, ovarian cancer, c-skin carcinoma. A positive signal was obtainedwith an undifferentiated lung sarcoma, but four other sarcoma sampleswere found to be negative.

c. Expression in malignant hemopathies:

summary:

acute myeloid leukemias: 6 positive samples (49 tested)

chronic myeloid leukemias: no positive samples (5 tested)

acute lymphoid leukemias: 11 positive samples (15 tested)

chronic lymphoid leukemias: no positive samples (2 tested)

multiple myeloma: no positive sample (1 tested)

Thus, the expression of the Oct-T1 gene is found in 11% of acute myeloidleukemia and 73% of acute lymphoid leukemia samples.

The results can also be presented in relation with the most frequentchromosomal abnormalities found in the acute leukemias:

t(9;22)(q34;q11): 1 positive sample (3 tested)

t(8;21)(q22;q22): 10 positive samples (11 tested)

t(3;21)(q26;q22): no positive samples (2 tested)

t(12;21)(p13;q22): 2 positive samples (2 tested)

Inv(16)(p13;q22): no positive samples (8 tested)

t(15;17)(q22;q21): no positive samples (4 tested)

11q23 rearrangement: no positive samples (3 tested)

trisomy 8: no positive samples (4 tested)

del 5/5q or del 7/7q: 3 positive samples (8 tested)

There is a clear correlation between the Oct-T1 gene expression and theacute leukemia with rearrangement of the AML1 gene, located on 21q22,and encoding the AML1 transcription factor.

4. Conclusion: The Oct-T1 gene is expressed in human acute leukemiacells. The gene is particularly frequently expressed in ALL. Theexpression of the gene is related to chromosomal rearrangementsinvolving the AML1 gene, which are involved in leukemogenesis.

Therefore, it may be possible to immunize leukemia patients againstantigens derived from the Oct-T1 protein in the form of peptidespresented by HLA class I molecules, and present at the surface ofleukemia cells expressing the gene. These antigens should not be presenton testis germinal cells, since these do not express HLA class Imolecules.

The Oct-T1 gene is expressed in leukemic cells, but not in normal bonemarrow, nor in normal PBL. Therefore, its specific and sensitivedetection by RT-PCR is potentially useful as a leukemia-specific tumormarker, for the detection of minimal residual disease, or for thequantitative evaluation of response to treatment after inductionchemotherapy.

Example 5 RT-PCR assavs for the expression of the preproTRH, thetryptase-L, and the Oct-T1/SIAX DP2-64 genes

Isolation of total RNA from tumor samples (quickly frozen at -80° C.)was performed by the guanidinium isothiocyanate/cesium chlorideprocedure (Davis et al., supra). cDNA synthesis was accomplished byextension with oligo(dT)₁₅. cDNA was then amplified by PCR with pairs ofoligonucleotide primers that are highly specific for each tested gene.To ensure that the RNA was not degraded, a PCR assay with primersspecific for β-actin was carried out.

1. cDNA synthesis The concentration of the RNA to be tested was adjustedto in 1 μl of total RNA/3.25 μl of water. The following reagents weremixed in a reaction tube placed in melting ice:

    ______________________________________    Reverse Transcriptase Buffer 5X                               4 μl    (Life Technologies Inc., Gaithersburg, MD)    dATP 10 mM                 1 μl    dCTP 10 mM                 1 μl    dGTP 10 mM                 1 μl    dTTP 10 mM                 1 μl    Dithiothreitol 100 mM      2 μl    oligo(dT).sub.15 20 μM  2 μl    Rnasin 40 units/μl (Promega Corp.)                               0.5 μl    M-MLV reverse transcriptase 200 units/μl    (Life Technologies, Inc.)  1 μl    Add 2 μg of template RNA                               6.5 μl    Total volume:              20 μl    ______________________________________

The reaction components were mixed and incubated at 42° C. for 60 min.The mixture was then chilled on ice. Water was added (80 μl) to obtain afinal volume of 100 μl. The mixture was store at -20° C. until used inPCR.

2. PCR amplification

a. Primers

    __________________________________________________________________________    preproTRH: sense primer (SEQ ID NO: 7): OPC376: 5'-CCAGCGGCTGCAAGGGGACCA-3       antisense primer (SEQ ID NO: 8): OPC377: 5'-TGCCCGCCGACCAGGGTGCT-3'    tryptase-L: sense primer (SEQ ID NO: 9): OPC314: 5'-CCCAAGAAGCCCTGAGC-3'       antisense primer (SEQ ID NO: 10): OPC315: 5'-CAAGAAAGGGGAGGGGG-3'    Oct-T1: sense primer (SEQ ID NO: 11): OPC406: 5'-CTGATCTAGTCCCAAGTCACC-3'    .       antisense primer (SEQ ID NO: 12): OPC407: 5'-ACAGCACTTGATCCAGAGTGG-3'    β-actin sense primer (SEQ ID NO: 13): OPC236: 5'-GGCATCGTGATGGACTCCG-    3'       antisense primer (SEQ ID NO: 14): OPC237: 5'-GCTGGAAGGTGGACAGCGA-3'    __________________________________________________________________________

b. PCR reaction: The following reagents were mixed in a reaction tubeplaced in melting ice:

H₂ O: 18.5 μl

PCR buffer 10× (Dynazyme): 2.5 μl

dNTP (10 mM each): 0.25 μl

sense primer (20 μM): 0.5 μl

antisense primer (20 μM): 0.5 μl

Dynazyme: 0.25 μl

cDNA was added (2.5 μl corresponding to 50 ng of total RNA), and thereaction mixture mixed. One drop of mineral oil (Sigma M-3516) waslayered on top of the PCR solution. The reaction tube was transferred tothe thermocycler for amplification. Positive control: cDNA fromSIAX/LB-1079; Negative control: water.

Thermal cycles: The PCR reactions were cycled as follows:

    ______________________________________    First denaturation:              94° for 5 min    Denaturation:              94° for 1 min    Annealing:    preproTRH 72° for 1 min 28 cycles for amplification of              preproTRH gene    tryptase-L              59° for 2 min 35 cycles for amplification of tryptase-L              gene    Oct-T1    63° for 2 min 27 cycles for amplification of Oct-T1              gene    β-actin              68° for 2 min 23 cycles for amplification of              β-actin              gene    Extension:              72° for 3 min    Final extension:              72° for 15 min    ______________________________________

The reactions were stored at 4° C. until used in agarose gelelectrophoresis.

3. Gel electrophoresis Aliquots (10 μl) of the PCR reaction wereelectrophoresed on a 1% agarose gel stained with ethidium bromide.

                                      TABLE 1    __________________________________________________________________________    GENE EXPRESSION IN MALIGNANT HEMOPATHIES    Code        Diagnosis               Karyotype           TRH                                      OCT-T1                                           Tryp-L    __________________________________________________________________________    CHIL        AML-M0 none                -  -    DRIA        AML-M0 46, XX, t(4;11)(q21;q23)                                   +/-                                      -    -    DUMA        AML-M0 46, XY              +  +    UMON        AML-M0 45, XX, t(9;22), -7 +  +++    CABU        AML-M1 47, XY, t(9;11)(p22;q23), +21                                   -  -    +    DEVA        AML-M1 46, XX              -  +/-    ELCA        AML-M1 46, XY, t(8;21)(q22;q22)                                   ++ +++  +    ETIT        AML-M1 46, XX, t(4;1;18)(q27;p35;q21), -2, -4,                                   -  -    GREM        AML-M1 46, XX              -  +/-    LENN        AML-M1 47, XX, +21         +  -    MELU        AML-M1 46, XX              -  -    KRIM        AML-M1 46, XY              -  -    BENA        AML-M1 46, XY              -  -    ALVA        AML-M2 42, XX, +8          -  -    -    AUWE        AML-M2 46, XY, -5, der(7)t(5;7)(q21;q2), +8                                   -  -    +++    BOUR        AML-M2 45, X, -Y, t(8;17;21)(q22;q11;q22)                                   ++ +++    DUWE        AML-M2 47, XY, t(9;22)(q34;q11),(+12), i(17q)                                   -  -    EVEL        AML-M2 46, XX              -  -    -    SIAX        AML-M2 46, XX, t(8;21)(q22;q22)                                   +++                                      +++    MARO        AML-M2 44, XX, -4, del(5)(q14;q31), -6, -7,                                   +/-                                      -    ENIS        AML-M2 46, XY, t(8;21)(q22;q22)                                   ++      +    RAMA        AML-M2 46, XX              +++                                      +++    LEMO        AML-M3 46, XY, t(15;17)(q22;q12)                                   +/-                                      -    KARA        AML-M3 46, XY, t(15;17)(q22;q12)                                   +/-                                      +/-    DEVI        AML-M3 46, XY, t(15;17)(q22;q12)                                   -  -    -    COWE        AML-M3 46, XX, t(15;17)(q22;q12)                                   +/-                                      -    LUSE        AML-M4 46, XX              -  -    REIB        AML-M4 46, XX              +/-                                      -    RICO        AML-M4 46, XY              -  -    AERT        AML-M4 46, XX              -  -    GIHA        AML-M4 46, XY              -  -    ERTE        AML-M4Eo               46, XX, inv(16)     +  -    TEEN        AML-M5 46, XY, t(8;11)(p22;q23)                                   -  -    DOMA        AML-M5 46, XX, del(7)(q22) +++                                      -    ROCH        AML-M5a                    -  -    DANA        AML-M5b               46, XY              -  -    LATT        AML-M5b               1) 46, XY, -5, ins(12;?)(p12;?), +M                                   -  -    LIAK        AML-M5b               1) 46, XX, inv(16)  -  -    +    OBBE        AML-M5b               46, XX              -  -    RYRO        AML-M5b               46,XX               -  -    LINT        AML-M5b               46, XY, inv(16)     +  -    RONI        AML-M5b               46, XX              -  -    TERE        AML-M5b               47, XY, t(2;7)(p1?4;p22), +8  10!                                   -  -    ERNI        AML-M5b               del 1q, trisomie 8  -  -    LOLI        AML-M5b               46, XY              -  -    NIES        AML-M6 44, XY, del(2)(q12;q14), del(7)(q32),                                   +/-                                      -    REUT        AML-M6 46, XX              +/-                                      -    GALU        AML-M6 44, XX, t(5;17)(p14;q11), -14, -20                                   +/-                                      -    OLBE        AML-M6 46, XX, t(21;21)    -  -    GRAD        CLL    46, XX              -  -    JEUM        CLL    46, XY              -  -    DERU        CML    46, XX, t(9;22)(q34;q11)                                   -  -    LOUY        CML    46, XY, t(9;22)     -  -    NEIR        CML    46, XY, t(9;22)(q34;q11)                                   -  -    VIER        CML    46, XY, t(9;22)     -  -    -    CAVA        ALL    1) 47, XY, t(9;22)(q34;q11), -9,                                   -  ++    AGUI        common ALL               1)47,XY,t(9;22)(q34;q11),-9                                   -  ++    DRON        common ALL               46, XX              +/-                                      -    GERN        common ALL               45, XY, -7, t(7;16)(q11.2;q24), -18, +mar                                   +  +    ISER        common ALL               46, XX, -7, del(12)(p11.?2), +mar                                   -  +    LULL        common ALL               47, X, der(X), del(1)(q25;q42), -3, t(6;22)                                   +/-                                      ++    MEUL        common ALL               47,XX,-2,der(2)t(2;9)(?p16;p23),del(12)(p13)                                   -  ++    SACK        common ALL               not known           -  -    WIRA        common ALL               46, XY              +  +    DANN        common ALL               46, XX              ++ +    -    POEL        common ALL               46, XY              +  ++    FURM        common ALL               46, XY, -18, +mar   +/-                                      ++    AMAY        common ALL                 -  -    QUEL        MM     81,XX,?add(5)(q3?2),-8,-9,-12,+15,-16,+22,                                   31 -    ORBA        MM    BETT        T-ALL  46, XY, del (7)(p13)                                   +/-                                      +    -    JASI        T-ALL  46, XX              -  +    OUSA        T-ALL  47, XY, +7          -  -    __________________________________________________________________________     N.B. results are expressed as relative intensity on the agarose gel, as     compared with positive control (SIAX), arbitrarily assigned +++ (+, ++,     +++ results are considered positive; -, +/- results are considered     negative).

Example 6 Identification of the portion of leukemia associated genesencoding a tumor rejection antigens.

In a first method, available CTL clones directed against antigenspresented by autologous tumor cells shown to express one or more of theleukemia associated genes are screened for specificity against COS cellstransfected with preproTRH, tryptase-L and/or Oct-T1 genes andautologous HLA alleles as described by Brichard et al. (Eur. J. Immunol.26:224-230, 1996). CTL recognition of preproTRH, tryptase-L and/orOct-T1 is determined by measuring release of TNF from the cytolytic Tlymphocyte or by ⁵¹ Cr release assay (Herin et al., Int. J. Cancer39:390-396, 1987). If a CTL clone specifically recognizes a transfectedCOS cell, shorter fragments of the coding sequences are prepared andtested by transfecting COS cells to identify the region of the gene thatencodes the peptide recognized by the CTL. Fragments of preproTRH,tryptase-L and/or Oct-T1 are prepared by exonuclease III digestion orother standard molecular biology methods such as PCR. Synthetic peptidesare prepared and tested to confirm the exact sequence of the antigen.

Alternatively, CTL clones are generated by stimulating the peripheralblood lymphocytes (PBLs) of a patient with autologous normal cellstransfected with DNA clones encoding preproTRH, tryptase-L and/or Oct-T1polypeptides (e.g. SEQ ID NOs:1, 3, 5 and/or 7) or with irradiated PBLsloaded with synthetic peptides corresponding to the putative proteinsand matching the consensus for the appropriate HLA class I molecule tolocalize the antigenic peptide within the preproTRH, tryptase-L and/orOct-T1 clones (see, e.g., van der Bruggen et al., Eur. J. Immunol.24:3038-3043, 1994; MAGE3 peptides presented by HLA.A2). The HLA type ofthe patient from which the leukemia cells from which preproTRH,tryptase-L and Oct-T1 were isolated is: A2, A26, B7, B56, Cw1, DR1, DR8,DQ4, DQ5.

Optionally, shorter fragments of preproTRH, tryptase-L and/or Oct-T1cDNAs are generated by PCR. Shorter fragments are used to provoke TNFrelease or ⁵¹ Cr release as above.

Example 7 Identification of leukemia associated gene encoded tumorrejection antigen peptides

Synthetic peptides corresponding to portions of the shortest fragment ofpreproTRH, tryptase-L and/or Oct-T1 which provokes TNF release areprepared. Progressively shorter peptides are synthesized to determinethe optimal preproTRH, tryptase-L and/or Oct-T1 tumor rejection antigenpeptides for a given HLA molecule.

Synthetic peptides are tested for lysis of HLA expressing cellsaccording to known procedures. For example, if the HLA which presents apeptide of interest is determined to be HLA-A2, then T2 cells can beused. T2 cells are HLA-A2⁺ cells which have an antigen-processing defectresulting in an increased capacity to present exogenous peptides. T2cells are mixed with a synthetic peptide corresponding to theCTL-reactive portion of preproTRH, tryptase-L or Oct-T1. CTL cells areadded and lysis is measured after 4 hours to determine which peptidesefficiently stimulate the lysis of T2 cells bearing HLA-A2. Other HLAexpressing cells are known in the art or can be prepared by transfectionwith specific HLA clones.

To determine the optimal size of the synthetic peptide, peptides ofdecreasing size are synthesized based on the sequence of the peptidedeterined above, by successively removing one amino acid from the aminoterminal end or the carboxy terminal end of the peptide. These peptidesare tested for the ability to induce cell lysis of appropriate HLAexpressing cells by CTL cells in a dose response assay. Lyophilizedpeptides are dissolved at 20 mg/ml in DMSO, then diluted to 2 mg/ml in10 mM acetic acid and stored at -80° C. Target cells, e.g. HLA-A2⁺ T2cells, are labeled with ⁵¹ Cr, as described above, for 1 hour at 37° C.followed by extensive washing to remove unincorporated label. To confirmthe necessity of the interaction of the peptide with the HLA, T2 cellsoptionally can be pretreated with an anti-HLA-A2 antibody, such as MA2.1(Wolfel et al., Eur. J. Immunol. 24: 759-764, 1994), and then areincubated in 96-well microplates in the presence of variousconcentrations of peptides for 30 minutes at 37° C. CTLs which recognizethe peptide presented by the HLA are then added in an equal volume ofmedium at an effector:target ratio of 30:1. Chromium-51 release ismeasured after 4 hours.

Example 8 Determination of the recognition of homologous peptides ofgenes related to leukemia associated genes by CTLs

As noted above, Oct-T1 and tryptase-L have high amino acid homology toother Oct family transcription factors and other tryptases. Todemonstrate that a tumor rejection antigen derived from Oct-T1 and/ortryptase-L is specific for these genes, peptides of other Oct andtryptase proteins which correspond to the postitions in the respectiveproteins of Oct-T1 and tyrptase-L are synthesized and used in a doseresponse-chromium release assay as described above. This experimentpermits the determination of the specificity of the Oct-T1 and/ortryptase-L derived TRAs, such that TRAs which selectively provoke lysisof cells which express Oct-T1 and/or tryptase-L, but not homologousgenes, can be selected.

Example 9 Normal cells are not lysed by CTLs which lyse cells whichexpress leukemia associated genes

This example describes CTL lysis experiments with various cell lineswith or without incubation with the leukemia associated gene derivedpeptides determined above. SIAX leukemic cells, normal B cells frompatient SIAX transformed with EBV (SIAX-EBV) and normal peripheral bloodlymphocytes from the same patient (SIAX-PBL) are tested for lysis by CTLcells in a dose response assay. These cells are incubated with CTLs atthe effector/target ratios determined to be optimal in the dose responseassays detailed above, and assayed for lysis as described above. Lysisof only the SIAX leukemic cells by the CTLs, demonstrates that SIAX-EBVand SIAX-PBL cells are not recognized by the CTLs because such cells donot normally express the tumor rejection antigen derived from preproTRH,tryptase-L and/or Oct-T1 proteins.

It is next determined whether these cells would be lysed by CTL ifpulsed with a peptide derived from preproTRH, tryptase-L and/or Oct-T1.The peptides selected on the basis of the experiments above are testedfor the ability to induce cell lysis of SIAX leukemic cells, SIAX-EBVcells, and non-autologous cells which express the appropriate HLA by CTLcells in a dose response assay as in previous examples. SIAX-EBV andSIAX-PBL pulsed with preferred peptides are not lysed by CTLs, but SIAXleukemic cells and the non-autologous cells pulsed with preferredpeptides are lysed by CTLs.

Other aspects of the invention will be clear to the skilled artisan andneed not be repeated here.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

A sequence listing is presented followed by what is claimed:

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 16    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1581 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 106..831    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CGCCCGGGGTCCTCAGCGCTGCAGACTCCTGACCTGCCGACTGCGGATCCCGAGTCCCCG60    GATCCCGGACCCATCCTGTGGAGCCCACTCCTGGCAGACGCCGCGATGCCCGGC114    MetProGly    CCTTGGTTGCTGCTCGCTCTGGCTTTGACCCTGAACCTGACCGGTGTC162    ProTrpLeuLeuLeuAlaLeuAlaLeuThrLeuAsnLeuThrGlyVal    51015    CCCGGCGGCCGTGCTCAGCCAGAGGCGGCCCAGCAGGAGGCAGTGACG210    ProGlyGlyArgAlaGlnProGluAlaAlaGlnGlnGluAlaValThr    20253035    GCCGCGGAGCATCCGGGCCTGGATGACTTCCTGCGCCAGGTGGAGCGC258    AlaAlaGluHisProGlyLeuAspAspPheLeuArgGlnValGluArg    404550    CTCCTCTTCCTCCGGGAAAACATCCAGCGGCTGCAAGGGGACCAGGGT306    LeuLeuPheLeuArgGluAsnIleGlnArgLeuGlnGlyAspGlnGly    556065    GAGCACTCCGCGTCCCAGATCTTTCAATCTGACTGGCTCTCCAAACGT354    GluHisSerAlaSerGlnIlePheGlnSerAspTrpLeuSerLysArg    707580    CAGCATCCAGGCAAAAGAGAGGAGGAGGAGGAAGAGGGAGTTGAAGAA402    GlnHisProGlyLysArgGluGluGluGluGluGluGlyValGluGlu    859095    GAGGAAGAGGAAGAAGGGGGGGCTGTGGGACCCCACAAACGGCAGCAC450    GluGluGluGluGluGlyGlyAlaValGlyProHisLysArgGlnHis    100105110115    CCTGGCCGACGAGAAGATGAGGCTTCATGGTCAGTCGATGTAACCCAG498    ProGlyArgArgGluAspGluAlaSerTrpSerValAspValThrGln    120125130    CACAAGCGGCAGCATCCTGGCCGGCGCTCCCCCTGGCTTGCATATGCT546    HisLysArgGlnHisProGlyArgArgSerProTrpLeuAlaTyrAla    135140145    GTCCCGAAGCGGCAGCACCCAGGCAGAAGGCTGGCAGATCCCAAGGCT594    ValProLysArgGlnHisProGlyArgArgLeuAlaAspProLysAla    150155160    CAAAGGAGCTGGGAAGAAGAGGAGGAGGAGGAAGAGAGAGAGGAAGAC642    GlnArgSerTrpGluGluGluGluGluGluGluGluArgGluGluAsp    165170175    CTGATGCCTGAAAAACGCCAGCATCCGGGCAAGAGGGCCCTGGGAGGC690    LeuMetProGluLysArgGlnHisProGlyLysArgAlaLeuGlyGly    180185190195    CCCTGTGGGCCCCAGGGAGCCTATGGTCAAGCGGGCCTCCTGCTGGGG738    ProCysGlyProGlnGlyAlaTyrGlyGlnAlaGlyLeuLeuLeuGly    200205210    CTCCTGGATGACCTGAGTAGGAGCCAGGGAGCTGAGGAAAAGCGGCAG786    LeuLeuAspAspLeuSerArgSerGlnGlyAlaGluGluLysArgGln    215220225    CACCCTGGTCGGCGGGCAGCCTGGGTCAGGGAGCCCCTGGAGGAG831    HisProGlyArgArgAlaAlaTrpValArgGluProLeuGluGlu    230235240    TGAACCCAGTTTTCCCTGAAGTCGAGTTTGTGGTCTAAGGATGTCTTGAGCCCTGTGTGC891    CCCACCATTCATGACCTCTGTATTCTCTAGTTAGATCCCTGACCATAAGCCTGAGCCCCT951    CCCTCCCAGCCCCATATTCACACACATCCCAGCCCCTGGCCTTGCCCTCTTCCTTTAGGC1011    ATGTGAGAAAATCAGCCTAGCAGTTTAAACCCCACTTTCCTCCACTTAGCACCATAGGCA1071    AGGGGGCAGATCCCAGAGCCCCTCTCACCCCCCCCACCACAGGCCTGCTCCTTCCTTAGC1131    CTTGGCTAAGATGGTCCTTCTGTGTCTTGCAAAGACTCCCCAAGTGGGACAGGGAGCCCC1191    TGGGAGGGCAGCCAGTGAGGGTGGGGTGGGACTGAAGCGTTGTGTGCAAATCCAGCTTCC1251    ATCCCCTCCCCAACCTGGCAGGATTCTCCATGTGTAAACTTCACCCCCAGGACCCAGGAT1311    CTTCTCCTTTCTGGGCATCCCTTTGTGGGTGGGCAGAGCCCTGACCCACAGCTGTGTTAC1371    TGCTTGGAGAAGCATATGTAGGGGCATACCCTGTGGTGTTGTGCTGTGTCTGGCTGTGGG1431    ATAAATGTGTGTGGGAATATTGAAACATCGCCTAGGAATTGTGGTTTGTATATAACCCTC1491    TAAGCCCCTATCCCTTGTCGATGACAGTCATCCTAATGATAATAAAACCTGCATCCAGAT1551    AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA1581    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 242 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetProGlyProTrpLeuLeuLeuAlaLeuAlaLeuThrLeuAsnLeu    151015    ThrGlyValProGlyGlyArgAlaGlnProGluAlaAlaGlnGlnGlu    202530    AlaValThrAlaAlaGluHisProGlyLeuAspAspPheLeuArgGln    354045    ValGluArgLeuLeuPheLeuArgGluAsnIleGlnArgLeuGlnGly    505560    AspGlnGlyGluHisSerAlaSerGlnIlePheGlnSerAspTrpLeu    65707580    SerLysArgGlnHisProGlyLysArgGluGluGluGluGluGluGly    859095    ValGluGluGluGluGluGluGluGlyGlyAlaValGlyProHisLys    100105110    ArgGlnHisProGlyArgArgGluAspGluAlaSerTrpSerValAsp    115120125    ValThrGlnHisLysArgGlnHisProGlyArgArgSerProTrpLeu    130135140    AlaTyrAlaValProLysArgGlnHisProGlyArgArgLeuAlaAsp    145150155160    ProLysAlaGlnArgSerTrpGluGluGluGluGluGluGluGluArg    165170175    GluGluAspLeuMetProGluLysArgGlnHisProGlyLysArgAla    180185190    LeuGlyGlyProCysGlyProGlnGlyAlaTyrGlyGlnAlaGlyLeu    195200205    LeuLeuGlyLeuLeuAspAspLeuSerArgSerGlnGlyAlaGluGlu    210215220    LysArgGlnHisProGlyArgArgAlaAlaTrpValArgGluProLeu    225230235240    GluGlu    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2259 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 8..577    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GGCCAGGATGCTGAGCCTGCTGCTGCTGGCGCTGCCCGTCCTGGCGAGC49    MetLeuSerLeuLeuLeuLeuAlaLeuProValLeuAlaSer    1510    CCGGCCTACGTGGCCCCTGCCCCAGGCCAGGCCCTGCAGCAAACGGGC97    ProAlaTyrValAlaProAlaProGlyGlnAlaLeuGlnGlnThrGly    15202530    ATTGTTGGGGGGCAGGAGGCCCCCAGGAGCAAGTGGCCCTGGCAGGTG145    IleValGlyGlyGlnGluAlaProArgSerLysTrpProTrpGlnVal    354045    AGCCTGAGAGTCCGCGGCCCATACTGGATGCACTTCTGCGGGGGCTCC193    SerLeuArgValArgGlyProTyrTrpMetHisPheCysGlyGlySer    505560    CTCATCCACCCCCAGTGGGTGCTAACCGCGGCGCACTGCGTGGAACCG241    LeuIleHisProGlnTrpValLeuThrAlaAlaHisCysValGluPro    657075    GACATCAAGGATCTGGCCGCCCTCAGGGTGCAACTGCGGGAGCAGCAC289    AspIleLysAspLeuAlaAlaLeuArgValGlnLeuArgGluGlnHis    808590    CTCTACTACCAGGACCAGCTGCTGCCGGTCAGCAGGATCATCGTGCAC337    LeuTyrTyrGlnAspGlnLeuLeuProValSerArgIleIleValHis    95100105110    CCACAGTTCTACATCATCCAGACCGGGGCGGACATCGCCCTGCTGGAG385    ProGlnPheTyrIleIleGlnThrGlyAlaAspIleAlaLeuLeuGlu    115120125    CTGGAGGAGCCCGTGAACATCTCCAGCCACATCCACACGGTCACGCTG433    LeuGluGluProValAsnIleSerSerHisIleHisThrValThrLeu    130135140    CCCCCTGCCTCGGAGACCTTCCCCCCGGGGATGCCGTGCTGGGTCACT481    ProProAlaSerGluThrPheProProGlyMetProCysTrpValThr    145150155    GGCTGGGGCGACGTGGACAATAATGGTGGGTGTTGGGGACAGCGGGAG529    GlyTrpGlyAspValAspAsnAsnGlyGlyCysTrpGlyGlnArgGlu    160165170    GCCGGGCCAGGTGGGCACCAAGTCACAGCCACAGGCCAGTCCGTGGGG577    AlaGlyProGlyGlyHisGlnValThrAlaThrGlyGlnSerValGly    175180185190    TGACAGGGTCCCTCAGGGCGGCTCAGGGAGGGGGACTGTGGAGGCCAGGATGGATGGAGC637    AGGCGGTGGCGAGAGGCAGCAGGTGCCCTGAGCAGAGACGGTGAGTCCAAAGGGCCTGGG697    CGTCCCCCACCCCAGGGGTTTGGAGAGTCCCTTAGCACCTCCGTGCCTCGGTTTCCCCTT757    GCCTGAAAGGGTGCATCAAAAGTTTGTACGTCACGGACTTGCTATGTGGAGAGAGAAATC817    ACACGGGGGTCTTGCTGGAAGGAGAGAGACCGGTGCTGGGATGAGACCTGCCTGCCCTCC877    ATCCCTGTGCTACAGACAAGGCAGGGGCCTGGGAATCGGGGTCGTGGCAGTGCTGTGGGG937    GGCTGGACGAAGCTCACTGTGGCCCTCCACGAGGCACATTTTCACTTCTAGAAGGTCTTG997    TCCCCATTTTATCCACAATTCAGAGCAAAGCTTTGGGGTACAGCCTGACGCAACCCTGGG1057    CTGTGACCTCTGGGTCACTCCAGAAGGGGCCTGAGCCACTGTCCCGCTATTCCGCCCCAC1117    ACAGCGGGGAAGCTGAGCCCAGCGCCCTGTGTTCCCCTCGGCTAGGGCCAACCGTGGACC1177    ATGGGCCTAGCCCAGACGAAAGTCAGCTGAGCCCAGGGGGAGACACGGGTCGGGCTCTGC1237    ACCCCCGTGCCATGGAGCCCAGCTTGGCAACCTCCAGGGCCCTCCCCTCCCTTCCCCAGA1297    TGGGGCTTAAATGAGGCCAGGGACCCAGGACCAGCCTCAGCGGAGGGGCCTGGACTGCAT1357    TCACCGCCCCTTCCCCGGGGCTGCAGGCACAGAACAGCACTGGGCCCATGGTGCCATCTC1417    CCCTGCCCGTGACTCTGCCACCAAGTCCACGAAGCAGCACCCAGCCGGCCCCAGACCCGG1477    CTCCACGCCCCCCTCCGCCCCCAGTGCACCTGCCGCCGCCATACCCGCTGAAGGAGGTGG1537    AAGTCCCCGTAGTGGAAAACCACCTTTGCAACGCGGAATATCACACCGGCCTCCATACGG1597    GCCACAGCTTTCAAATCGTCCGCGATGACATGCTGTGTGCGGGGAGCGAAAATCACGACT1657    CCTGCCAGGGTGACTCTGGAGGGCCCCTGGTCTGCAAGGTGAATGGCACCTAACTGCAGG1717    CGGCCGTGGTCAGCTGGGAGGAGAGCTGTGCCCAGCCCAACCGGCCTGGCATCTACACCC1777    GTGTCACCTACTACTTGGACTGGATCCACCACTATGTCCCCAAGAAGCCCTGACCCAGGC1837    CTGGGTTGTCCACCCGGGTCACTGGAGGGCCAGCCCCTCCTGTCCAAACCACCACTGCTT1897    CCTACCCAGGTGGTGACTAAACCCCACACCTTCCCCCATCCTGAGTCCCCTCTCCCATCC1957    TGAGCCCTGTCCCCTGTCCTGAGCCCCCTCCCCTTTCTTGATCCCCTCCCCCATCCTGAG2017    CCCCCTCCCCCACCCTGAGCCCCCTCCCCTGTCTTGAGCCCCTCATCCATCCTGAGCCCC2077    TCCTCCATCCTGAGCCCCCTCCCCCATCCTGGGCCCCCTCCCCTTTCTTGAGCCCCCTCC2137    CCCACCCTGAGCCCCTTCCCCTTTCTTGAGCCCCTCCTCCACCCTCAGCCCCCTCCCCTT2197    TCTTGAGCCCCTCCTCCACCCTCAGCCCCCTCCCCTTTCTTGAGCCCCTCCCCCACCCTG2257    AG2259    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 190 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetLeuSerLeuLeuLeuLeuAlaLeuProValLeuAlaSerProAla    151015    TyrValAlaProAlaProGlyGlnAlaLeuGlnGlnThrGlyIleVal    202530    GlyGlyGlnGluAlaProArgSerLysTrpProTrpGlnValSerLeu    354045    ArgValArgGlyProTyrTrpMetHisPheCysGlyGlySerLeuIle    505560    HisProGlnTrpValLeuThrAlaAlaHisCysValGluProAspIle    65707580    LysAspLeuAlaAlaLeuArgValGlnLeuArgGluGlnHisLeuTyr    859095    TyrGlnAspGlnLeuLeuProValSerArgIleIleValHisProGln    100105110    PheTyrIleIleGlnThrGlyAlaAspIleAlaLeuLeuGluLeuGlu    115120125    GluProValAsnIleSerSerHisIleHisThrValThrLeuProPro    130135140    AlaSerGluThrPheProProGlyMetProCysTrpValThrGlyTrp    145150155160    GlyAspValAspAsnAsnGlyGlyCysTrpGlyGlnArgGluAlaGly    165170175    ProGlyGlyHisGlnValThrAlaThrGlyGlnSerValGly    180185190    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2218 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: NVB352/3    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 8..577    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GGCCAGGATGCTGAGCCTGCTGCTGCTGGCGCTGCCCGTCCTGGCGAGC49    MetLeuSerLeuLeuLeuLeuAlaLeuProValLeuAlaSer    1510    CCGGCCTACGTGGCCCCTGCCCCAGGCCAGGCCCTGCAGCAAACGGGC97    ProAlaTyrValAlaProAlaProGlyGlnAlaLeuGlnGlnThrGly    15202530    ATTGTTGGGGGGCAGGAGGCCCCCAGGAGCAAGTGGCCCTGGCAGGTG145    IleValGlyGlyGlnGluAlaProArgSerLysTrpProTrpGlnVal    354045    AGCCTGAGAGTCCGCGGCCCATACTGGATGCACTTCTGCGGGGGCTCC193    SerLeuArgValArgGlyProTyrTrpMetHisPheCysGlyGlySer    505560    TTCATCCACCCCCAGTGGGTGCTAACCGCGGCGCACTGCGTGGAACCG241    PheIleHisProGlnTrpValLeuThrAlaAlaHisCysValGluPro    657075    GACATCAAGGATCTGGCCGCCCTCAGGGTGCAACTGCGGGAGCAGCAC289    AspIleLysAspLeuAlaAlaLeuArgValGlnLeuArgGluGlnHis    808590    CTCTACTACCAGGACCAGCTGCTGCCGGTCAGCAGGATCATCGTGCAC337    LeuTyrTyrGlnAspGlnLeuLeuProValSerArgIleIleValHis    95100105110    CCACAGTTCTACATCATCCAGACCGGGGCGGACATCGCCCTGCTGGAG385    ProGlnPheTyrIleIleGlnThrGlyAlaAspIleAlaLeuLeuGlu    115120125    CTGGAGGAGCCCGTGAACATCTCCAGCCACATCCACACGGTCACGCTG433    LeuGluGluProValAsnIleSerSerHisIleHisThrValThrLeu    130135140    CCCCCTGCCTCGGAGACCTTCCCCCCGGGGATGCCGTGCTGGGTCACT481    ProProAlaSerGluThrPheProProGlyMetProCysTrpValThr    145150155    GGCTGGGGCGACGTGGACAATAATGGTGGGTGTTGGGGACAGCGGGAG529    GlyTrpGlyAspValAspAsnAsnGlyGlyCysTrpGlyGlnArgGlu    160165170    GCCGGGCCAGGTGGGCACCAAGTCACAGCCACAGGCCAGTCCGTGGGG577    AlaGlyProGlyGlyHisGlnValThrAlaThrGlyGlnSerValGly    175180185190    TGACAGGGTCCCTCAGGGCGGCTCAGGGAGGGGGACTGTGGAGGCCAGGATGGATGGAGC637    AGGCGGTGGCGAGAGGCAGCAGGTGCCCTGAGCAGAGACGGTGAGTCCAAAGGGCCTGGG697    CGTCCCCCACCCCAGGGGTTTGGAGAGTCCCTTAGCACCTCCGTGCCTCGGTTTCCCCTT757    GCCTGAAAGGGTGCATCAAAAGTTTGTACGTCACGGACTTGCTATGTGGAGAGAGAAATC817    ACACGGGGGTCTTGCTGGAAGGAGAGAGATCGGTGCTGGGATGAGACCTGCCTGCCCTCC877    ATCCCTGTGCTACAGACAAGGCAGGGGCCTGGGAATCGGGGTCGTGGCAGTGCTGTGGGG937    GGCTGGACGAAGCTCACTGTGGCCCTCCACGAGGCACATTTTCACTTCTAGAAGGTCTTG997    TCCCCATTTTATCCACAATTCAGAGCAAAGCTTTGGGGTACAGCCTGAGCGGCAACCCTG1057    GGCTGTGACTCTGGGTCACTCAGAAGGGGCCTGAGCCACTGTCCCGCTATTCCGCCCCAC1117    ACAGCGGGGAAGCTGAGCCCAGCGCCCTGTGTTCCCCTCGGCTAGGGCCAACCGTGGACC1177    ATGGGCCTAGCCCAGACGAAAGTCAGCTGAGCCCAGGGGGAGACACGGGTCGGGCTCTGC1237    ACCCCCGTGCCATGGAGCCCAGCTTGGCAACCTCCAGGGCCCTCCCCTCCCTTCCCCAGA1297    TGGGGCTTAAATGAGGCCAGGGACCCAGGACCAGCCTCAGCGGAGGGGCCTGGACTGCAT1357    TCACCGCCCCTTCCCCGGGGCTGCAGGCACAGAACAGCACTGGGCCCATGGTGCCATCTC1417    CCCTGCCCGTGACTCTGCCACCAAGTCCACGAAGCAGCACCCAGCCGGCCCCAGACCCGG1477    CTCCACGCCCCCCTCCGCCCCCAGTGCACCTGCCGCCGCCATACCCGCTGAAGGAGGTGG1537    AAGTCCCCGTAGTGGAAAACCACCTTTGCAACGCGGAATATCACACCGGCCTCCATACGG1597    GCCACAGCTTTCAAATCGTCCGCGATGACATGCTGTGTGCGGGGAGCGAAAATCACGACT1657    CCTGCCAGGTGGGCCCTCGCGTCCCCCACCCCAATCCCCGGAGCCTGGCCAGCGAGCGCA1717    TCCCTCATCCTGACCCCCGAAGCCTGGCCAGCGAGCACTGACCTCTGACCTTCCCAGGGT1777    GACTCTGGAGGGCCCCTGGTCTGCAAGGTGAATGGCACCTAACTGCAGGCGGGCGTGGTC1837    AGCTGGGAGGAGAGCTGTGCCCAGCCCAACCGGCCTGGCATCTACACCCGTGTCACCTAC1897    TACTTGGACTGGATCCACCACTATGTCCCCAAGAAGCCCTGAGCCAGGCCTGGGGTGTCC1957    ACCCGGGTCACTGGAGGGCCAGCCCCTCCTGTCCAAACCACCACTGCTTCCTACCCAGGT2017    GGTGACTGCCCCCCACACCTTCCCCCATCCTGAGTCCCCTCTCCCATCCTGAGCCCTGTC2077    CCCTGTCCTGAGCCCCCTCCCCTTTCTTGATCCCCTCCCCCATCCTGAGCCCCCTCCCCC2137    ACCCTGAGCCCCCTCCCCTGTCTTGAGCCCCTGCTCCATCCTGAGTCCCCTCCCCCACAC2197    TGAGCCCCCTCCCCTTTCTTG2218    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 190 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    MetLeuSerLeuLeuLeuLeuAlaLeuProValLeuAlaSerProAla    151015    TyrValAlaProAlaProGlyGlnAlaLeuGlnGlnThrGlyIleVal    202530    GlyGlyGlnGluAlaProArgSerLysTrpProTrpGlnValSerLeu    354045    ArgValArgGlyProTyrTrpMetHisPheCysGlyGlySerPheIle    505560    HisProGlnTrpValLeuThrAlaAlaHisCysValGluProAspIle    65707580    LysAspLeuAlaAlaLeuArgValGlnLeuArgGluGlnHisLeuTyr    859095    TyrGlnAspGlnLeuLeuProValSerArgIleIleValHisProGln    100105110    PheTyrIleIleGlnThrGlyAlaAspIleAlaLeuLeuGluLeuGlu    115120125    GluProValAsnIleSerSerHisIleHisThrValThrLeuProPro    130135140    AlaSerGluThrPheProProGlyMetProCysTrpValThrGlyTrp    145150155160    GlyAspValAspAsnAsnGlyGlyCysTrpGlyGlnArgGluAlaGly    165170175    ProGlyGlyHisGlnValThrAlaThrGlyGlnSerValGly    180185190    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4524 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 174..1433    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    CAGAGGGAGCGCCTGGCAGCAGCAGGAGCAGCAGCAGCAGCCCGCGGCGGGGCCGCCGCC60    AGCCGCCGCGACCGCCGCGGCTGCAGCCTCCGAAGGGAGGCCGGGTGAGCCGGCGTACGC120    ACTTTCCCGCGGACTTTCGGAGTGTTTGTGGATATACATGCCAAGCCGCCACGATG176    Met    1    ATGTCCATGAACAGCAAGCAGCCTCACTTTGCCATGCATCCCACCCTC224    MetSerMetAsnSerLysGlnProHisPheAlaMetHisProThrLeu    51015    CCTGAGCACAAGTACCCGTCGCTGCACTCCAGCTCCGAGGCCATCCGG272    ProGluHisLysTyrProSerLeuHisSerSerSerGluAlaIleArg    202530    CGGGCCTGCCTGCCCACGCCGCCGCTGCAGAGCAACCTCTTCGCCAGC320    ArgAlaCysLeuProThrProProLeuGlnSerAsnLeuPheAlaSer    354045    CTGGACGAGACGCTGCTGGCGCGGGCCGAGGCGCTGGCGGCCGTGGAC368    LeuAspGluThrLeuLeuAlaArgAlaGluAlaLeuAlaAlaValAsp    50556065    ATCGCCGTGTCCCAGGGCAAGAGCCATCCTTTCAAGCCGGACGCCACG416    IleAlaValSerGlnGlyLysSerHisProPheLysProAspAlaThr    707580    TACCACACGATGAACAGCGTGCCGTGCACGTCCACTTCCACGGTGCCT464    TyrHisThrMetAsnSerValProCysThrSerThrSerThrValPro    859095    CTGGCGCACCACCACCACCACCACCACCACCACCAGGCGCTCGAACCC512    LeuAlaHisHisHisHisHisHisHisHisHisGlnAlaLeuGluPro    100105110    GGCGATCTGCTGGACCACATCTCCTCGCCGTCGCTCGCGCTCATGGCC560    GlyAspLeuLeuAspHisIleSerSerProSerLeuAlaLeuMetAla    115120125    GGCGCGGGCGGCGCGGGCGCGGCGGCCGGCGGCGGCGGCGCCCACGAC608    GlyAlaGlyGlyAlaGlyAlaAlaAlaGlyGlyGlyGlyAlaHisAsp    130135140145    GGCCCGGGGGGCGGTGGCGGCCCGGGCGGCGGCGGCGGCCCGGGCGGC656    GlyProGlyGlyGlyGlyGlyProGlyGlyGlyGlyGlyProGlyGly    150155160    GGCGGCCCCGGGGGAGGCGGCGGTGGCGGCCCGGGGGGCGGCGGCGGC704    GlyGlyProGlyGlyGlyGlyGlyGlyGlyProGlyGlyGlyGlyGly    165170175    GGCCCGGGCGGCGGGCTCCTGGGCGGCTCCGCGCACCCTCACCCGCAT752    GlyProGlyGlyGlyLeuLeuGlyGlySerAlaHisProHisProHis    180185190    ATGCACAGCCTGGGCCACCTGTCGCACCCCGCGGCGGCGGCCGCCATG800    MetHisSerLeuGlyHisLeuSerHisProAlaAlaAlaAlaAlaMet    195200205    AACATGCCGTCCGGGCTGCCGCACCCCGGGCTGGTGGCGGCGGCGGCG848    AsnMetProSerGlyLeuProHisProGlyLeuValAlaAlaAlaAla    210215220225    CACCACGGCGCGGCAGCGGCAGCGGCGGCGGCGTCGGCCGGGCAGGTG896    HisHisGlyAlaAlaAlaAlaAlaAlaAlaAlaSerAlaGlyGlnVal    230235240    GCAGCGGCATCGGCGGCGGCGGCCGTGGTGGGCGCAGCGGGCCTGGCG944    AlaAlaAlaSerAlaAlaAlaAlaValValGlyAlaAlaGlyLeuAla    245250255    TCCATCTGCGACTCGGACACGGACCCGCGCGAGCTCGAGGCGTTCGCG992    SerIleCysAspSerAspThrAspProArgGluLeuGluAlaPheAla    260265270    GAGCGCTTCAAGCAGCGGCGCATCAAGCTGGGCGTGACGCAGGCCGAC1040    GluArgPheLysGlnArgArgIleLysLeuGlyValThrGlnAlaAsp    275280285    GTGGGCTCGGCGCTGGCCAACCTCAAGATCCCGGGCGTGGGCTCACTC1088    ValGlySerAlaLeuAlaAsnLeuLysIleProGlyValGlySerLeu    290295300305    AGCCAGAGCACCATCTGCAGGTTCGAGTCGCTCACGCTCTCGCACAAC1136    SerGlnSerThrIleCysArgPheGluSerLeuThrLeuSerHisAsn    310315320    AACATGATCGCGCTCAAGCCCATCCTGCAGGCGTGGCTCGAGGAGGCC1184    AsnMetIleAlaLeuLysProIleLeuGlnAlaTrpLeuGluGluAla    325330335    GAGGGCGCCCAGCGCGAGAAAATGAACAAGCCTGAGCTCTTCAACGGC1232    GluGlyAlaGlnArgGluLysMetAsnLysProGluLeuPheAsnGly    340345350    GGCGAGAAGAAGCGCAAGCGGACTTCCATCGCCGCGCCCGAGAAGCGC1280    GlyGluLysLysArgLysArgThrSerIleAlaAlaProGluLysArg    355360365    TCCCTCGAGGCCTACTTCGCCGTGCAGCCCCGGCCCTCGTCCGAGAAG1328    SerLeuGluAlaTyrPheAlaValGlnProArgProSerSerGluLys    370375380385    ATCGCCGCCATCGCCGAGAAACTGGACCTCAAAAAGAACGTGGTGCGG1376    IleAlaAlaIleAlaGluLysLeuAspLeuLysLysAsnValValArg    390395400    GTGTGGTTTTGCAACCAGAGACAGAAGCAGAAGCGGATGAAATTCTCT1424    ValTrpPheCysAsnGlnArgGlnLysGlnLysArgMetLysPheSer    405410415    GCCACTTACTGAGGGGGCTGGGAGGTGTCGGGCGGGACAGAATGGGGAG1473    AlaThrTyr    420    CTGAGGAGGCATTTTTGGGGGGCTTTCCTCTGCTTGCCTCCCCTCGGATTTGGAGTGTCC1533    GTTATCCTGCCTGCATTTGGGGAGTCCCTTCTCGCTCTCTTTCCTCCACCCATTCTCTGA1593    TTTTCCTGCCTTTGCTGTCCCCTAGCCTTGAGGACTGGGGTGCTGGGTGTGGGGATTGGA1653    GTATAGGGTAGGGGAGAAGGGGGGGAGCATTCGGGGGAGTGGGGAGTGGGGGGAAGGAAA1713    GCGGAGACCCGAGCAGGGGTTTTAAGGAGCAGGATGGTTCTGGGGTTTGGGTGGGGGGAG1773    ACGCGGGAAGGGTAGGAAAATGGACTGTTTCTGACCAGAGACACTTACCTAAATATCCTG1833    GGGACCAAGGAACTATGTACAAAAACAAACCTACCAACCACCAAAAACTAGACAAATAAA1893    GACAAACTAAAACAAAACAGAACAAAAGCAAAGGAAAATGCTTTAGAAATTTTAACTCCG1953    GGGAGCCATAATCTGCAACTTCATTTTCCCCCATAGAAGAGAAAAAAGAGCACCACCATT2013    ATTACCACCTCCCCAACCCTACACGCACGAACTGAGTCGAAAAACGAAAACCAAACGAGC2073    GAGAAGTTGAAGTTCTGGGTATCAAAGCTAGTTGTTCTGTCTGCGTGTTTAATTTTTCCC2133    TCTCTCACCTCCACCCCATCCATATCCTCTTTATTTCCTCCGTTCCAATGAGAGGCCTAT2193    GGCTGCTCTCCAATCCCGGGAAGTGAGTGGGAGCACAGCTGAAAAGAGAGGGTCAGGGGG2253    AGGCTGGCTGCTTGCTTAGGTGGAATCCAAGTTTTCCCGTGGCCCTGCCTATACTCTGGT2313    GGCCTGGTCCTGTTGGGGTGGGGGTCTTTGGAGAGAAGGGCATAGTCTTTGAGCTACTAA2373    AAAGCAGAATTCCGGAGCTTCGAGATATCTTATTCTAGGAAAATGAAACAATTTTAACAA2433    CAGTTTTTTTTCCTCTTATGTCGAAGATCTAGTTTTAGACAATTTCAAAATAAGCTTTTC2493    CCACTCATAGAACTTTAACTTGCCCTTTCAGTTTTATCTTTTTTTTAGAGAGAGGTTTAA2553    ACTACTGATTTTTCCTGTTGATTCAAATAGACTAATGGGGTGAAAGTTATTAGGAGAGAT2613    ACTCTCTCCTGTTTTCTCCACTGAACGAGACTCATCTTGCTCTTCTAGGTCCCGTTTCTT2673    CCTCTCTTGGAGGACATGAAATTATAGAAATGTTGAGAAGTTCCTGCTTTCTTTTGCGGT2733    AGGACTTGGCTGTGAGAAAATCACCTAAATCCCAGAAAAGAGGAAGACAGATTTAAAGTG2793    CCCCCACCCCCATTTGTTTCAAAGAGGTCTGCATGTTGGGCGAAAACAGAACAACTGTGT2853    TTCCTTTTACTTGTTCTTATTATTCAAGAGTCATTTATTACAGGGGATAAATGTTGGGTA2913    GCAAGAACTTTAATTTGCACTACCAGTCTCCCAAATAGAAAATCATGTATAGTATTTCAT2973    AGTAATAATCAGGTACCTTACAAGCTGCTGGTGGATTTTAAAAAATTAAGATAGTTGAAG3033    GTGGTTAGGTAAAATGCCTGCTTTGTGTACAAGATACTCTTTGGATCTCTCGTAGAGATG3093    GTTTGTTACCATCCTTTAATCATAACTAAAACATTGAAAACAGAACAAATGAGAAAAGAA3153    AAAAAACCTGCCGATTAACAAGACTGAAATCATGCATGATCTGAAAGGTGTGGAAAGAAA3213    CACAATTAGGTCTCACTCTGGTTAGGCATTATTTATTTAATTATGTTGTATATCATTGTT3273    TGCAGGGCAAACATTCTATGCATTTGAAACTGAGCACTAAACTGGGCTAGCTTTCTGGTA3333    GACCGTTTTGTGGCTAGTGCGATTTCACAGTCTACTGCCTGTTTCCACTGAAAACATTTT3393    TGTCATATTCTTGTATTCAAAGAAAAAGGAAAAAAGATTATTGTAAATATTTTATTTAAT3453    GCACACATTCACACAGTGGTAACAGACTGCCAGTGTTCATCCTGAAATGTCTCACGGATT3513    GATCTACCTGTCCATGTATGTCTGCTGAGCTTTCTCCTTGGTTATGTTTTTTCTCTTTTA3573    CCTTTCTCCTCCCTTACTTCTATCAGAACCAATTCTATGCGCCAAAATACAACAGGGGGA3633    TGTGTCCCAGTACACTTACAAATAAAACATAACTGAAAGAAGAGCAGTTTTATGATTTGG3693    GTGCGTTTTTGTGTTTATACTGGGCCAGGTCCTGGTAGAACCTTTCAACAAACAACCAAA3753    CAAAAAGAAAACACAAAGAAATGGGGGGAGGGTAGGGTGTTGAAGGGGGACAAAAAGGGG3813    AGAGATTGAGAATGATGTATTTTTTTGCTGAATCAGAATTCACTTTCAGATAACTCATGA3873    AAAGTGGTGCTCTAAATAAAATGAATTCTATATTAGTTGCCTGTGTTTATAAAAGTTATT3933    ATTTTTTAACTGCAGAAACTCTTAAACCACAGCACTTGATCCAGAGTGGTGAAAACCAAT3993    AAATAACCAGGCACCCAAAAAAACATTTAAATTTAGGGTCAGGGACAGAGGAATTTGGAG4053    GTTTAGATGTGATATTCTACCCTAAAAACACCTAGTAACTGAATGGCTTTTCTTTGGAGG4113    GTAATACATTTTAAAACATTTAGTGTGCCACCTACTGCTCCACAGTGACTAGAGAGCCTC4173    TATTTCTTGGTGACTTGGGACTAGATCAGATGCCAAATGTACAAAGTTTCTTAAGAGTTG4233    AGATTATATCATCTGAAGTCATCTTATTTTAGCCAAATCTTTTTAATTTCACCGGCAAAT4293    CTGTGAAGGAAAACACTTGATGTTCAAAAAGAATAGTACATTTTAAAAGCTGCGATTTTA4353    AACAGTTGTTAATGTTAAAAAAAAAAAGCACTAGAGGTATTTTTAAACATAGAACTCTTC4413    CATAAAAAGTTGATTTGTTTTTGCTGTTATTGACTTGAAACATCATCAGTTTTAAATAAA4473    ATGCATTTGTAAAAAAACCGTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAA4524    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 420 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    MetMetSerMetAsnSerLysGlnProHisPheAlaMetHisProThr    151015    LeuProGluHisLysTyrProSerLeuHisSerSerSerGluAlaIle    202530    ArgArgAlaCysLeuProThrProProLeuGlnSerAsnLeuPheAla    354045    SerLeuAspGluThrLeuLeuAlaArgAlaGluAlaLeuAlaAlaVal    505560    AspIleAlaValSerGlnGlyLysSerHisProPheLysProAspAla    65707580    ThrTyrHisThrMetAsnSerValProCysThrSerThrSerThrVal    859095    ProLeuAlaHisHisHisHisHisHisHisHisHisGlnAlaLeuGlu    100105110    ProGlyAspLeuLeuAspHisIleSerSerProSerLeuAlaLeuMet    115120125    AlaGlyAlaGlyGlyAlaGlyAlaAlaAlaGlyGlyGlyGlyAlaHis    130135140    AspGlyProGlyGlyGlyGlyGlyProGlyGlyGlyGlyGlyProGly    145150155160    GlyGlyGlyProGlyGlyGlyGlyGlyGlyGlyProGlyGlyGlyGly    165170175    GlyGlyProGlyGlyGlyLeuLeuGlyGlySerAlaHisProHisPro    180185190    HisMetHisSerLeuGlyHisLeuSerHisProAlaAlaAlaAlaAla    195200205    MetAsnMetProSerGlyLeuProHisProGlyLeuValAlaAlaAla    210215220    AlaHisHisGlyAlaAlaAlaAlaAlaAlaAlaAlaSerAlaGlyGln    225230235240    ValAlaAlaAlaSerAlaAlaAlaAlaValValGlyAlaAlaGlyLeu    245250255    AlaSerIleCysAspSerAspThrAspProArgGluLeuGluAlaPhe    260265270    AlaGluArgPheLysGlnArgArgIleLysLeuGlyValThrGlnAla    275280285    AspValGlySerAlaLeuAlaAsnLeuLysIleProGlyValGlySer    290295300    LeuSerGlnSerThrIleCysArgPheGluSerLeuThrLeuSerHis    305310315320    AsnAsnMetIleAlaLeuLysProIleLeuGlnAlaTrpLeuGluGlu    325330335    AlaGluGlyAlaGlnArgGluLysMetAsnLysProGluLeuPheAsn    340345350    GlyGlyGluLysLysArgLysArgThrSerIleAlaAlaProGluLys    355360365    ArgSerLeuGluAlaTyrPheAlaValGlnProArgProSerSerGlu    370375380    LysIleAlaAlaIleAlaGluLysLeuAspLeuLysLysAsnValVal    385390395400    ArgValTrpPheCysAsnGlnArgGlnLysGlnLysArgMetLysPhe    405410415    SerAlaThrTyr    420    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    CCAGCGGCTGCAAGGGGACCA21    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    TGCCCGCCGACCAGGGTGCT20    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    CCCAAGAAGCCCTGAGC17    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    CCCTCAAGAAAGGGGAGGGGG21    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    CTGATCTAGTCCCAAGTCACC21    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    ACAGCACTTGATCCAGAGTGG21    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GGCATCGTGATGGACTCCG19    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    GCTGGAAGGTGGACAGCGA19    __________________________________________________________________________

We claim:
 1. A method for diagnosing a disorder characterized byexpression of a leukemia associated nucleic acid molecule,comprising:contacting a biological sample isolated from a subject withan agent that is specific for the leukemia associated nucleic acidmolecule, wherein the leukemia associated nucleic acid moleculehybridizes under stringent conditions to a molecule having a nucleotidesequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5 and SEQ ID NO:7, and determining the interaction between theagent and the leukemia associated nucleic acid molecule as adetermination of the disorder.
 2. The method of claim 1 wherein theagent is a nucleic acid molecule comprising a molecule having anucleotide sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, fragments thereof, andcomplements thereof.
 3. The method of claim 1, wherein the agentcomprises a nucleic acid molecule having the nucleotide sequence of SEQID NO:1 or a fragment thereof.
 4. The method of claim 3, wherein thebiological sample is isolated from a non-fetal-brain tissue.
 5. Themethod of claim 1, wherein the agent comprises a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:5 or afragment thereof.
 6. The method of claim 5, wherein the biologicalsample is isolated from a non-mastocyte tissue.
 7. The method of claim1, wherein the agent comprises a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:7 or a fragment thereof.
 8. The methodof claim 7, wherein the biological sample is isolated from anon-fetal-testis tissue.
 9. The method of claim 1 wherein theinteraction is determined by amplifying at least a portion of thenucleic acid molecule.